U.S. patent number 11,092,592 [Application Number 16/776,855] was granted by the patent office on 2021-08-17 for unmasking endotoxins in solution.
This patent grant is currently assigned to BIOMERIEUX DEUTSCHLAND GMBH. The grantee listed for this patent is BIOMERIEUX DEUTSCHLAND GMBH. Invention is credited to Bernd Buchberger.
United States Patent |
11,092,592 |
Buchberger |
August 17, 2021 |
**Please see images for:
( Certificate of Correction ) ** |
Unmasking endotoxins in solution
Abstract
The invention relates to unmasking endotoxins in compositions so
that previously present, but undetectable endotoxins are rendered
detectable.
Inventors: |
Buchberger; Bernd
(Zeitlarn/Laub, DE) |
Applicant: |
Name |
City |
State |
Country |
Type |
BIOMERIEUX DEUTSCHLAND GMBH |
Nurtingen |
N/A |
DE |
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Assignee: |
BIOMERIEUX DEUTSCHLAND GMBH
(Nurtingen, DE)
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Family
ID: |
51059260 |
Appl.
No.: |
16/776,855 |
Filed: |
January 30, 2020 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20200166500 A1 |
May 28, 2020 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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15316884 |
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10585086 |
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PCT/EP2015/063152 |
Jun 12, 2015 |
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62011868 |
Jun 13, 2014 |
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Foreign Application Priority Data
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Jun 12, 2014 [EP] |
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14172158 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N
33/15 (20130101); G01N 33/56911 (20130101); G01N
33/52 (20130101) |
Current International
Class: |
G01N
33/52 (20060101); G01N 33/15 (20060101); G01N
33/569 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0308239 |
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Mar 1989 |
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EP |
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0 322 786 |
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Jul 1989 |
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EP |
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1917976 |
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May 2008 |
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EP |
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425216 |
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Mar 1935 |
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GB |
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H01-156910 |
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Jun 1989 |
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JP |
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H01-294629 |
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Nov 1989 |
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JP |
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H07-255462 |
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Oct 1995 |
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JP |
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2005-530991 |
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Oct 2005 |
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JP |
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2006-194606 |
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Jul 2006 |
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JP |
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2008-514771 |
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May 2008 |
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JP |
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2009-155613 |
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Jul 2009 |
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JP |
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WO 2002/057789 |
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Jul 2002 |
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WO |
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WO 2006/129662 |
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May 2006 |
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WO |
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WO 2009/152384 |
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Dec 2009 |
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WO |
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Other References
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|
Primary Examiner: Gangle; Brian
Attorney, Agent or Firm: Parker Highlander PLLC
Parent Case Text
This application is a divisional of U.S. patent application Ser.
No. 15/316,884, filed Dec. 7, 2016, which is a national phase
application under 35 U.S.C. .sctn. 371 of International Application
No. PCT/EP2015/063152, filed Jun. 12, 2015, which claims the
benefit of U.S. Provisional Patent Application Ser. No. 62/011,868,
filed Jun. 13, 2014, and claims the benefit of European Patent
Application No. 14172158.9, filed Jun. 12, 2014. The entirety of
each of the above-referenced disclosures are incorporated herein by
reference.
Claims
The invention claimed is:
1. An aqueous composition comprising a protein, an aliphatic
compound with C8-C16 as the main chain, and lipopolysaccaride
(LPS), wherein the aliphatic compound is an alkanol, and wherein
the protein is chosen from an antibody, an antibody fragment, a
hormone, an enzyme, a fusion protein, and any combination
thereof.
2. The aqueous composition according to claim 1, wherein the
alkanol is an unbranched 1-alkanol.
3. The aqueous composition according to claim 1, wherein the
alkanol is a branched compound with at least one substitution in
the main chain selected from a methyl, ethyl, propyl and butyl
group.
4. The aqueous composition according to claim 1, further comprising
a detergent selected from an anionic detergent, a cationic
detergent, a nonionic detergent, an amphoteric detergent and any
combination thereof.
5. The aqueous composition according to claim 4, wherein said
detergent is an anionic detergent chosen from the group consisting
of: alkyl sulfates; alkyl-ether sulfates; cholesterol sulfate;
sulfonates; alkyl sulfo succinates; sulfoxides; phosphates; and
carboxylates.
6. The aqueous composition according to claim 4, wherein said
detergent is a cationic detergent chosen from the group consisting
of: primary amines; secondary amines; tertiary amines; and
quaternary ammonium cations; cetylpyridinium chloride (CPC);
quaternary ammonium detergents; and hydroxyethylcellulose
ethoxylate, quaternized (Polyquaternium-10).
7. The aqueous composition according to claim 4, wherein said
detergent is a nonionic detergent chosen from the group consisting
of: polyoxyethylene glycol sorbitan alkyl esters (polysorbates);
polyoxyethylene glycol alkyl ethers; polyoxypropylene glycol alkyl
ethers; glucoside alkyl ethers; polyoxyethylene glycol octylphenol
ethers; polyoxyethylene glycol alkylphenol ethers; glycerol alkyl
esters; sorbitan alkyl esters; block copolymers of polyethylene
glycol and polypropylene glycol; cocamide MEA; sterols;
cyclodextrins; poloxamers; and cocamide DEA.
8. The aqueous composition according to claim 4, wherein said
detergent is an amphoteric detergent chosen from the group
consisting of: CHAPS
(3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate);
sultaines; betaines; amino oxides; and lecithin.
9. The aqueous composition according to claim 4, wherein the
detergent is selected from Polysorbate 20, Polysorbate 80,
Poloxamer 188, Octoxynol 9, Laurylamineoxid,
tris[2-(2-hydroxyethoxy)ethyl]-octadecyl-ammonium phosphate,
trilaureth-4 phosphate and sodium stearate.
10. The aqueous composition according to claim 1, wherein the
antibody fragment is selected from a Fab, a Fab', a F(ab')2 and an
Fv, a single chain antibody and any combination thereof.
11. The aqueous composition according to claim 1, containing a
further protein which is an albumin.
12. The aqueous composition according to claim 1, further
comprising a chaotropic agent, a cation or a combination
thereof.
13. The aqueous composition according to claim 12, wherein the
composition comprises a chaotropic agent selected from urea,
guanidinium chloride, butanol, ethanol, lithium perchlorate,
lithium acetate, magnesium chloride, phenol, propanol and
thiourea.
14. The aqueous composition according to claim 12, wherein the
composition comprises a cation that is a divalent cation.
15. The aqueous composition according to claim 14, wherein the
divalent cation is selected from Ca2+, Mg2+, Sr2+ and Zn2+.
16. The aqueous composition according to claim 11, wherein the
further protein is present in a concentration from 0.1-20 mg/ml;
the aliphatic compound is present in the concentration from
0.01-100 mM; the detergent is present in a concentration from
0.001-1.0 wt %; and/or the divalent cation is present in the
concentration from 1-400 mM.
17. The aqueous composition according to claim 16, further
comprising a chaotropic agent in a concentration from 1 mM-1 M.
18. The aqueous composition according to claim 1, wherein the pH is
in the range from pH 2-12.
19. The aqueous composition according to claim 1, further
containing Factor C protein.
20. The aqueous composition according to claim 2, wherein the
unbranched 1-alkanol is 1-dodecanol.
21. The aqueous composition according to claim 5, wherein said
detergent is ammonium lauryl sulfate or sodium lauryl sulfate
(SDS).
22. The aqueous composition according to claim 5, wherein said
detergent is sodium laureth sulfate or sodium myreth sulfate.
23. The aqueous composition according to claim 5, wherein said
detergent is dodecylbenzensulfonate, sodium lauryl sulfoacetate or
xylene sulfonate.
24. The aqueous composition of claim 5, wherein the detergent is
disodium lauryl sulfosuccinate.
25. The aqueous composition according to claim 5, wherein said
detergent is dodecyl methyl sulfoxide or trilaureth-4
phosphate.
26. The aqueous composition according to claim 5, wherein said
detergent is sodium stearate or sodium lauroyl sarcosinate.
27. The aqueous composition according to claim 6, wherein said
detergent is an alkyltrimethylammonium salt.
28. The aqueous composition according to claim 27, wherein said
detergent is cetyl trimethylammonium bromide (CTAB) or cetyl
trimethylammonium chloride (CTAC).
29. The aqueous composition according to claim 6, wherein said
detergent is tris[2-(2-hydroxyethoxy)ethyl]-octadecyl-ammonium
phosphate.
30. The aqueous composition according to claim 7, wherein said
detergent is polysorbate 20, polysorbate 40, polysorbate 60 or
polysorbate 80.
31. The aqueous composition according to claim 7, wherein said
detergent is cholesterol.
32. The aqueous composition according to claim 7, wherein said
detergent is a poloxamer block polymer.
33. The aqueous composition according to claim 8, wherein said
detergent is cocamidopropyl hydroxysultaine or cocamidopropyl
betaine.
34. The aqueous composition according to claim 8, wherein said
detergent is palmitamine oxide, laurylamine oxide or amine oxide of
general formula R.sup.3N.sup.+O.sup.-, wherein R.sup.3 is
C.sub.8-C.sub.18 alkyl, C.sub.8-C.sub.18 alkenyl, or
C.sub.8-C.sub.18 alkynyl.
35. The aqueous composition according to claim 11, wherein the
further protein is human serum albumin, bovine serum albumin and/or
ovalbumin.
36. The aqueous composition according to claim 16, wherein the
further protein is present in a concentration from 1-10 mg/ml; the
aliphatic compound is present in the concentration from 0.1-10 mM;
the detergent is present in a concentration from 0.05-0.5 wt %;
and/or the divalent cation is present in the concentration from
10-200 mM.
37. The aqueous composition according to claim 19, wherein the
Factor C protein is a recombinant Factor C protein.
Description
The present invention relates to unmasking endotoxins in
compositions, preferably pharmaceutical compositions, so that
present but undetectable endotoxins are rendered detectable.
Specifically, the invention relates to a method of unmasking an
endotoxin in a composition. The invention further relates to a
method of detecting an endotoxin in a composition. The invention
further relates to a kit for unmasking an endotoxin in a
composition. The invention further relates to the use of a
modulator capable of unmasking an endotoxin, e.g. by releasing an
endotoxin from a complex between said endotoxin and an endotoxin
masker, to unmask an endotoxin in a composition.
BACKGROUND OF THE INVENTION
Endotoxins are part of the outer membrane of the cell wall of
Gram-negative bacteria. Endotoxin is invariably associated with
Gram-negative bacteria regardless of whether the organisms are
pathogenic or not. Although the term "endotoxin" is occasionally
used to refer to any cell-associated bacterial toxin, in
bacteriology it is properly reserved to refer to the
lipopolysaccharide (LPS) complex associated with the outer membrane
of Gram-negative pathogens such as Escherichia coli, Salmonella,
Shigella, Pseudomonas, Neisseria, Haemophilus influenzae,
Bordetella pertussis and Vibrio cholerae.
The presence of endotoxins in aqueous compositions is an
intractable problem which severely threatens and/or limits the
application of many compositions, in particular if intended for
pharmaceutical use. This is especially true of compositions
comprising protein products, e.g. recombinant protein products.
Naturally occurring endotoxins, especially endotoxins belonging to
the class of compounds characterized as lipopolysaccharides (LPS)
are molecules produced by certain types of bacteria, for example
gram-negative bacteria. Generally, endotoxins such as LPS comprise
an extended polysaccharide O-antigen, a core antigen polysaccharide
including an outer core component and an inner core component, and
a lipid A domain comprising aliphatic amides and aliphatic acid
esters. Such endotoxins are found in the outer membrane of
gram-negative bacteria, where they contribute to bacterial
structural integrity by shielding the organism from chemical
attack. Such endotoxins increase the negative charge of the cell
membrane of these bacteria, and help to stabilize the overall
membrane structure. Such endotoxins elicit strong responses from
normal animal, e.g. human, immune systems because normal serum
contains lipooligosaccharide (LOS) receptors which normally direct
the cytotoxic effects of the immune system against invading
bacterial pathogens bearing such endotoxins.
When present in the human blood in a form disassociated from their
source bacteria, endotoxins such as LPS can cause endotoxemia which
in severe cases can lead to septic shock. This reaction is due to
the endotoxin lipid A component, which can cause uncontrolled
activation of the mammalian immune system, in some instances
producing inflammatory mediators such as toll-like receptor (TLR)
4, which is responsible for immune system cell activation.
Bacteria, as well as the endotoxins they produce, are also
ubiquitous. For instance, endotoxin contaminants are known to exist
in the pipes and hoses of water supply systems, including those of
laboratories and facilities for preparing pharmaceutical
formulations. The surfaces of containers such as fermentors and
glassware used in the process of formulating pharmaceuticals are
also commonly contaminated. In addition, as humans carry bacteria
and therefore endotoxins on their bodies, so the staff of such
facilities in which pharmaceuticals are formulated also represent a
possible source of endotoxin contaminants.
Of course, in addition to the above, gram-negative bacteria
themselves find wide use in the production of i.a. recombinant
therapeutic proteins, so there is always a danger that endotoxin
contamination of aqueous compositions, e.g. pharmaceutical
formulations, containing such therapeutic proteins may also arise
directly from such bacteria used in the production process.
To safeguard against potentially hazardous incorporation of
endotoxin contaminants, whatever their source, measures must
normally be taken to exclude endotoxin from all steps and products
used in the production process of such proteins before such
solutions may be administered for therapeutic purposes. In fact,
the exclusion and/or removal and verifiable absence of all traces
of (detectable) endotoxin are among the requirements which much
must be met when seeking regulatory approval for any new
therapeutic, in particular those containing products produced in
bacteria, or which have come into contact with bacteria at any
point in the production process (see e.g. EMEA, Q6B,
Specifications: Test Procedures and Acceptance Criteria for
Biotechnological/Biological Products; 2.1.4 Purity, Impurities and
Contaminants; Contaminants; 4.1.3 Purity and impurities; 2) FDA,
Q6B, Specifications: Test Procedures and Acceptance Criteria for
Biotechnological/Biological Products; II.A.4. Purity, Impurities
and Contaminants; IV.A.3. Purity and Impurities). For instance, all
containers holding and/or transferring solutions intended for
eventual administration must be rendered endotoxin-free prior to
contact with the solution. A depyrogenation oven is used for this
purpose, in which temperatures in excess of 200.degree. C. are
required to break down endotoxins. Based on primary packaging
material as syringes or vials, a glass temperature of 250.degree.
C. and a holding time of 30 minutes is typical to achieve a
reduction of endotoxin levels by a factor of 1000. Usually, liquids
cannot be depyrogenated by heat, therefore different methods are
used, such as chromatography (e.g. anion exchange), phase
extraction (e.g. Trition X-114), filtration (e.g.
ultrafiltration).
One common assay for detecting the activity of endotoxin is the
limulus amebocyte lysate (LAL) assay, which utilizes blood from the
horseshoe crab. Very low levels of endotoxin can cause coagulation
by the limulus lysate due to a powerful amplification through an
enzyme cascade. However, due to the dwindling population of
horseshoe crabs, efforts have been made to develop alternative,
e.g. recombinant, Factor C assays for detecting the presence of
endotoxin in solution. The most promising of such methods are
enzyme-linked affinitysorbent assays, using a solid phase for
endotoxin capturing and subsequent detection by recombinant version
of a protein in the LAL assay, Factor C. The EndoLISA.RTM. kit is
one such affinitysorbent assay.
However, even the best available tests for detecting the presence
of pyrogens, such as endotoxin, in particular LPS, are often unable
to detect LPS in solution. This implies the danger that solutions
which are reasonably--in the absence of any detectable
endotoxin--thought to be endotoxin-free in fact contain endotoxin
which is simply masked so as to be rendered undetectable. Such
solutions, e.g. pharmaceutical formulations will not be barred from
regulatory approval (at least not due to containing endotoxin),
because by all diagnostic appearances, these solutions are
endotoxin-free, therefore fulfilling--or at least appearing to
fulfill--this regulatory requirement. Clearly, however,
administration of such ostensibly endotoxin-free solutions to
subjects risks triggering the types of reactions mentioned above.
In such instances, one may learn of the presence of masked
endotoxin in such solutions too late, after subjects have already
developed the types of adverse and potentially life-threatening
reactions described above. In addition, from a hygenic standpoint,
drug regulatory authorities place great value on positively knowing
which substances are contained in pharmaceutical compositions and
which are not. This ultimately comes down to the ability to
reliably detect all components in a given composition, and one's
ability to believe the results obtained in reference to both the
presence and absence of all substances tested.
It should be noted that the terms "masking" and "unmasking", as
pertain to endotoxins, have been used with various meanings in the
literature. On the one hand, the literature uses the term
"endotoxin unmasking" or "endotoxin demasking" to describe removal
of endotoxin from certain solutions (e.g. protein solutions). In
this case, a certain endotoxin content is detectable before and
after using common procedures for endotoxin removal (e.g.
chromatography). Where the available techniques are inadequate for
complete removal of endotoxin from the particular sample, the
endotoxin which cannot be removed is referred to as "masked"
endotoxin; any endotoxin which can be removed by available
techniques is referred to as "unmasked" or "demasked" endotoxin.
According to this usage of the term, "masked" endotoxin thus
denotes endotoxin which cannot be removed, and implies insufficient
removal of (detectable) endotoxin.
On the other hand, the literature also uses the term "endotoxin
masking" in the case of inadequate endotoxin detection. In this
case, only a fractional amount or, in many cases, no endotoxin
whatsoever can be detected, although endotoxin is present.
According to this usage of the term, "masked" endotoxin thus
denotes endotoxin which cannot be detected, or can only barely be
detected, and implies insufficient endotoxin detection.
Inadequate detection of endotoxin can occur in various
compositions. For example in protein solutions (Petsch et al.,
Analytical Biochemistry 259, 42-47, 1998), drug products (J. Chen
and K. Williams, Follow-Up on Low Endotoxin Recovery in Biologics
PDA Letter, October 2013), or even in common formulation components
of drug products (J. Reich et al., Poster: Low Endotoxin Recovery
in Common Protein Formulations, 6th Workshop on Monoclonal
Antibodies, Basel, Switzerland, 2013; J. Reich et al., Poster: Low
Endotoxin Recovery in Biologics: Case Study--Comparison of Natural
Occurring Endotoxin (NOE) and Commercially Available Standard
Endotoxin, PDA Annual meeting, San Antonio, USA, 2014).
WO 2009/152384 A1 discloses notional compositions by defining
categories of components in the compositions and then providing
lists of components within each category. This document does not
disclose any individualized composition comprising a protein, a
C8-C16 alkanol and LPS.
Similarly, WO 02/057789 A2 discloses notional compositions by
defining categories of components in the compositions and then
providing lists of components within each category. This document
does not disclose any individualized composition comprising a
protein, a C8-C16 alkanol and LPS.
EP 1 917 976 A1 discloses certain compositions, but does not
disclose any composition comprising a protein, a C8-C16 alkanol and
LPS.
There thus exists a strong motivation to provide ways in which all
endotoxin present in compositions, including endotoxin which is
undetectable because it is being masked by certain other
composition components, may be unmasked such that it is rendered
detectable. Providing a way to unmask and/or detect hitherto
undetectable endotoxin in a composition would greatly assist in
promoting patient safety. It is an aim of the present invention to
address such needs.
BRIEF SUMMARY OF THE INVENTION
The present invention relates to an aqueous composition comprising
a protein and an aliphatic compound with C.sub.8-C.sub.16 as the
main chain and which compound preferably has a substitution by one
or more heteroatoms.
The aqueous composition may preferably be a pharmaceutical
composition containing a protein to which the aliphatic compound is
added. The addition of the aliphatic compound helps to improve the
detectability of a potential contamination of the composition by an
LPS. As stated in other parts of this application, LPS might escape
detection by conventional endotoxin tests because of being masked
by some constituents of protein-containing compositions.
According to a preferred embodiment, the aliphatic compound is a
branched compound with at least one substitution in the main chain
wherein the substitution may be selected from methyl, ethyl, propyl
and butyl groups.
The main chain of the aliphatic compound is as defined elsewhere
herein.
According to a further preferred embodiment, the main chain is
selected from a C.sub.8-C.sub.16 alkyl, C.sub.8-C.sub.16 alkenyl
and C.sub.8-C.sub.16 alkynyl. The main chain may contain one or
more double bonds and/or one or more triple bonds, whereas a
saturated alkyl chain is the more preferred embodiment.
According to a further preferred embodiment, the heteroatom that
may form part of the aliphatic compound is selected from O, S and
N, whereas O is the more preferred substitution.
A further preferred aliphatic compound is selected from an alkanol,
which is preferably an unbranched alkanol, more preferably a
1-alkanol and most preferably 1-dodecanol.
The aliphatic compound is assumed to stabilize a potentially
contaminating LPS molecule in a form that renders LPS more
susceptible to detection by conventional endotoxin test kits such
as the EndoLISA.RTM. by Hyglos GmbH.
Compositions that might be rendered more susceptible to the
detection of endotoxin often contain detergents which may be
selected from an anionic detergent, a cationic detergent, a
nonionic detergent, an amphoteric detergent and any combination
thereof. Preferred detergents that may be used in such compositions
may be selected from: an anionic detergent which can be chosen from
the group consisting of: alkyl sulfates, preferably ammonium lauryl
sulfate or sodium lauryl sulfate (SDS); alkyl-ether sulfates,
preferably sodium laureth sulfate or sodium myreth sulfate;
cholesterol sulfate; sulfonates, preferably dodecylbenzensulfonate,
sodiumlauryl sulfoacetate or xylene sulfonate; alkyl sulfo
succinates, preferably disodium lauryl sulfosuccinate; sulfoxides,
preferably dodecyl methyl sulfoxide; phosphates, preferably
trilaureth-4 phosphate; and carboxylates, preferably sodium
stearate or sodium lauroyl sarcosinate;
a cationic detergent which can be chosen from the group consisting
of: primary amines; secondary amines; tertiary amines; and
quaternary ammonium cations such as alkyltrimethylammonium salts
(preferably cetyl trimethylammonium bromide (CTAB); or cetyl
trimethylammonium chloride (CTAC)); cetylpyridinium chloride (CPC);
quaternary ammonium detergents, preferably
tris[2-(2-hydroxyethoxy)ethyl]-octadecyl-ammonium phosphate
(Quaternium 52); and hydroxyethylcellulose ethoxylate, quaternized
(Polyquaternium-10);
a nonionic detergent which can be chosen from the group consisting
of: polyoxyethylene glycol sorbitan alkyl esters (polysorbates),
preferably polysorbate 20 (TWEEN.TM.-20), polysorbate 40,
polysorbate 60 or polysorbate 80 (TWEEN.TM.-80); polyoxyethylene
glycol alkyl ethers; polyoxypropylene glycol alkyl ethers;
glucoside alkyl ethers; polyoxyethylene glycol octylphenol ethers;
polyoxyethylene glycol alkylphenol ethers; glycerol alkyl esters;
sorbitan alkyl esters; block copolymers of polyethylene glycol and
polypropylene glycol; cocamide MEA; sterols, preferably
cholesterol; cyclodextrins; poloxamers, preferably Pluronic block
polymers; and cocamide DEA;
an amphoteric detergent which can be chosen from the group
consisting of: CHAPS
(3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate);
sultaines, preferably cocamidopropyl hydroxysultaine; betaines,
preferably cocamidopropyl betaine; amino oxides, preferably
palmitamine oxide, laurylamine oxide and amine oxide of general
formula R.sup.3N.sup.+O.sup.-, wherein R.sup.3 is C.sub.8-C.sub.18
alkyl, C.sub.8-C.sub.18 alkenyl, or C.sub.8-C.sub.18 alkynyl; and
lecithin.
According to a further preferred embodiment, the detergent is
selected from a polysorbate, preferably Polysorbate 20 and
Polysorbate 80, a poloxamer, preferably Poloxamer 188, an
octoxynol, preferably an Octoxynol 9, an alkylamine oxide,
preferably laurylamine oxide, a quaternary ammonium salt,
preferably tris[2-(2-hydroxyethoxy)ethyl]-octadecyl-ammonium
phosphate, an alkyl phosphate, preferably trilaureth-4 phosphate,
and a stearate, preferably sodium stearate.
In a preferred aqueous composition, the protein is chosen from an
antibody, an antibody fragment, a hormone, an enzyme, a fusion
protein, a protein conjugate and any combination thereof, which
proteins are frequently used as the active agent of pharmaceutical
preparations where specific care must be taken that LPS does not
remain undetected in the quality control of pharmaceuticals.
In a further preferred embodiment, the antibody fragment is
selected from a Fab, a Fab', a F(ab')2 and an Fv, a single chain
antibody and any combination thereof.
In a further preferred embodiment, the aqueous composition, in
addition to the active pharmaceutical ingredient, which may be the
protein mentioned above, may contain an additional protein selected
from an albumin, which is preferably human serum albumin, bovine
serum albumin and/or ovalbumin. The further protein may be of
assistance in rendering a potential LPS contamination more
detectable by conventional endotoxin tests such as the ones
mentioned above.
In a further preferred embodiment, the aqueous composition may
comprise a chaotropic agent, a cation or a combination thereof. The
same ingredients also can help to bring a potential LPS
contamination into a form that is more susceptible to detection by
an endotoxin test by Hyglos GmbH.
According to a further preferred embodiment, the chaotropic agent
is selected from urea, guanidinium chloride, butanol, ethanol,
lithium perchlorate, lithium acetate, magnesium chloride, phenol,
propanol and thiourea.
According to a further preferred embodiment, the cation is a
divalent cation, preferably selected from Ca2+, Mg2+, Sr2+ and
Zn2+.
According to a further preferred embodiment, the further protein,
which may be an albumin, is present in a concentration in the range
from 0.1-20 mg/ml, preferably in the range from 1-10 mg/ml, more
preferably in an amount of 10 mg/ml.
In a further preferred embodiment, the aliphatic compound is
present in the concentration from 0.01-100 mM, preferably in a
concentration from 0.1-10 mM. This concentration range is in
particular preferred for an 1-alkanol, preferably 1-dodecanol.
In a further preferred embodiment, the detergent is present in a
concentration from 0.001-1.0 wt %, preferably 0.05-0.5 wt %,
preferably from 0.02-0.2 wt %.
In a further preferred embodiment, the chaotopic agent is present
in a concentration from 1 mM-1 M, preferably from 25-200 mM,
preferably from 10 mM-100 mM.
In a further preferred embodiment, the divalent cation is present
in a concentration from 1-400 mM, preferably in a concentration
from 10-200 mM, more preferably in a concentration from 50-100
mM.
In a further preferred embodiment, the pH of the composition is in
the range from 2-12, preferably in the range from pH 5-10.
In a further preferred embodiment, the composition contains Factor
C protein, which is a component used in for conventional endotoxin
assays.
In a preferred embodiment, the Factor C protein is a recombinant
Factor C protein.
A very preferred aqueous composition comprises a protein,
preferably an antibody, in combination with a 1-alkanol, preferably
1-dodecanol in a concentration range from 0.1-10 mM, a detergent of
claim 8 in a concentration range from 0.002-0.2 wt %, a divalent
cation, preferably Ca2+, in a concentration range from 10-200 mM,
and a pH from 5 to 10.
A further very preferred aqueous composition is as set out above in
the immediately preceding paragraph, and further comprising a
chaotropic agent, preferably guanidinium chloride, in the
concentration range from 10 mM-100 mM.
In the above compositions, LPS, if present, will be susceptible to
detection by conventional endotoxin assays such as the EndoLisa of
Hyglos GmbH.
One disclosure relates to a method of unmasking an endotoxin in a
composition, preferably a pharmaceutical composition, comprising an
endotoxin masker and suspected of comprising said endotoxin, said
method comprising the step of adding to said composition a
modulator capable unmasking said endotoxin, e.g. by of releasing
said endotoxin, if present, from a complex between said endotoxin
and said endotoxin masker. The pharmaceutical composition will in
most cases be an aqueous composition.
A further disclosure relates to a method of detecting an endotoxin
in a composition, preferably a pharmaceutical composition,
comprising an endotoxin masker and suspected of comprising said
endotoxin, said method comprising the steps of adding to said
composition a modulator capable of unmasking said endotoxin, e.g.
by releasing said endotoxin, if present, from a complex between
said endotoxin and said endotoxin masker; and detecting said
endotoxin by means of a detection method. The pharmaceutical
composition will in most cases be an aqueous composition.
In certain embodiments, the above methods of unmasking and/or
detecting may further comprise the step of adding to said
composition an agent which influences hydrogen bonding stability in
solution. In certain embodiments, it is preferable to add said
agent which influences hydrogen bonding stability in solution to
said solution prior to the addition of said modulator.
A further disclosure relates to a kit for unmasking an endotoxin in
a composition, preferably a pharmaceutical composition, comprising
an endotoxin masker and suspected of comprising said endotoxin,
said kit comprising a) a modulator capable of unmasking said
endotoxin, e.g. by releasing said endotoxin from a complex between
said endotoxin and said endotoxin masker; and b) an agent which
influences hydrogen bonding stability in solution; wherein
components (a) and (b) are in same or different packages.
A further disclosure relates to a use of a modulator capable of
unmasking endotoxin, e.g. by releasing an endotoxin from a complex
between said endotoxin and an endotoxin masker, to unmask an
endotoxin in a composition, preferably a pharmaceutical composition
suspected of comprising said endotoxin and said endotoxin
masker.
Other embodiments of this invention will be readily apparent from
the following disclosure.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 illustrates a mechanism assumed to underlie the unmasking of
endotoxin according to an embodiment of the present invention. In
the scenario depicted in FIG. 1, the endotoxin is present in
solution with a detergent (capable of acting as an endotoxin
masker), which forms detergent micelles in which endotoxin is
embedded and thus masked from detection. FIG. 1 schematically shows
the effects of adding a single-component modulator which breaks up
these micelles, liberating embedded endotoxin, while not forming
new micelles of its own. Following breakup of the detergent
micelles, the single-component modulator then serves as a chaperone
to the liberated endotoxin, stabilizing it in solution. An
equilibrium exists between individual and aggregated endotoxin
moieties, and the detection of the endotoxin aggregate proceeds
based on the aggregated form ("Aggregates are the biologically
active units of endotoxins". Mueller, M., Lindner, B., Kusomoto,
S., Fukase, K., Schromm, A. B. and Seydel, U. (2004) The Journal of
Biological Chemistry, Vol. 279, No. 25, pp. 26307-26313. Endotoxin
in the form shown in panel (a) is not susceptible to detection,
whereas endotoxin in the form shown in panel (c) is detectable. The
scenario depicted in FIG. 1 is discussed in further detail
below.
FIG. 2 illustrates a mechanism assumed to underlie the unmasking of
endotoxin according to a further embodiment of the present
invention. In the scenario depicted in FIG. 2, the endotoxin is
present in solution with a detergent (capable of acting as an
endotoxin masker), which forms detergent micelles in which
endotoxin is embedded and thus masked from detection. FIG. 2
schematically shows the effects of adding a dual-component
modulator comprising protein and non-protein components. This
dual-component modulator is assumed to break apart the detergent
micelle in which the endotoxin was previously inserted and masked.
The non-protein component of the modulator transiently stabilizes
the endotoxin outside of the detergent micelle, while the protein
component of the modulator destabilizes the detergent micelle by
binding i.a. molecules of detergent. The scenario depicted in FIG.
2 is discussed in further detail below.
FIG. 3 illustrates a mechanism assumed to underlie the unmasking of
endotoxin according to a further embodiment of the present
invention. In the scenario depicted in FIG. 3, the endotoxin is
present in solution with a detergent (capable of acting as an
endotoxin masker), which forms detergent micelles in which
endotoxin is embedded and thus masked from detection. FIG. 3
schematically shows the effects of adding a multiple-component
modulator, as well as an agent influencing hydrogen bonding
stability. Together, the multiple-component modulator and the agent
influencing hydrogen bonding stability destabilize the detergent
micelle initially masking the endotoxin, and promote endotoxin
aggregation such that it is rendered detectable. The scenario
depicted in FIG. 3 is discussed in further detail below.
FIG. 4 illustrates a mechanism assumed to underlie the unmasking of
endotoxin according to a further embodiment of the present
invention. In the scenario depicted in FIG. 4, the endotoxin is
present in solution, i.a. with a protein. The protein comprises a
binding cleft in which endotoxin may stably bind and thus remain
masked from detection. FIG. 4 schematically shows the effects of
adding a multiple-component modulator such that the previously
masked endotoxin aggregates and is rendered detectable. The
scenario depicted in FIG. 4 is discussed in further detail
below.
FIG. 5 illustrates a mechanism assumed to underlie the unmasking of
endotoxin according to a further embodiment of the present
invention. In the scenario depicted in FIG. 5, the endotoxin is
present in solution with a protein (capable of acting as an
endotoxin masker). The protein comprises a binding cleft in which
endotoxin may stably bind and thus remain masked from detection.
FIG. 5 schematically shows the effects of adding an agent
influencing hydrogen bonding stability as well as a
multiple-component modulator including protein and non-protein
components. Together, these destabilize the endotoxin in its
complex with the masking protein, transiently stabilize endotoxin
outside of the complex with the masking protein, and promote
aggregation of the liberated endotoxin, rendering it detectable.
The scenario depicted in FIG. 5 is discussed in further detail
below.
FIG. 6 illustrates a mechanism assumed to underlie the unmasking of
endotoxin according to a further embodiment of the present
invention. In the scenario depicted in FIG. 6, the endotoxin is
present in solution with a protein as well as with a detergent
(capable of acting as an endotoxin masker). The protein comprises a
binding cleft in which endotoxin may stably bind and thus remain
masked from detection. In addition, the detergent forms stable
micelles in which molecules of endotoxin stably inserted are
masked. FIG. 6 schematically shows the effects of adding an agent
influencing hydrogen bonding stability as well as a
multiple-component modulator including protein and non-protein
components. Together, these destabilize the endotoxin in its
complex with the masking protein and/or in the masking detergent
micelle, transiently stabilize endotoxin outside of the complex
with the masking protein and/or in the masking detergent micelle,
and promote aggregation of the liberated endotoxin, rendering it
detectable. The scenario depicted in FIG. 6 is discussed in further
detail below.
FIG. 7 is a graph showing the percent recovery of the endotoxin LPS
from a detergent masker (polysorbate 20/citrate) using modulator
systems of 1-dodecanol alone, and 1-dodecanol together with
BSA.
FIG. 8 is a graph showing the percent recovery of the endotoxin LPS
from the detergent masker TRITON.TM. (non-ionic surfactant) X-100
using various modulator systems of various strengths.
FIG. 9 is a graph showing the percent recovery of the endotoxin LPS
from various detergent masking systems using a variety of modulator
systems.
FIG. 10 is a graph showing the percent recovery of the endotoxin
LPS from a masking detergent (polysorbate 20) as dependent on
pH.
FIG. 11 is a graph showing the percent recovery of the endotoxin
LPS from a masking detergent (polysorbate 80) as dependent on
pH.
FIG. 12 is a flowchart showing a generalized validation scheme for
determining and optimizing an unmasking process for a composition
in question suspected of containing masked endotoxin.
FIG. 13 is a table showing a generalized evaluation scheme for
determining and optimizing an unmasking process for a composition
in question suspected of containing masked endotoxin.
FIG. 14 is a general schematic representation of the inventive
methods herein, as viewed from the standpoint of the level of LPS
recovery (i.e. measured LPS activity) before and after masking
(left and middle bars of figure, respectively), as well as after
unmasking according to the methods of the present invention (right
bar of figure). The left and middle bars of the figure thus
represent the circumstances commonly prevailing in pharmaceutical
formulations, in which endotoxin which is present in solution, is
rendered undetectable by one or more endotoxin maskers. This
endotoxin can be again rendered detectable, i.e. can be "rescued"
out of its masked state, by the methods of the present invention,
enabling one to detect the previously masked endotoxin.
FIG. 15 shows a generic diagram illustrating the dynamics
associated with the unmasking methods described herein. The
transition from active (i.e. aggregated and therefore detectable)
LPS at the far left to masked LPS (middle bottom; non-aggregated)
is shown for several representative endotoxins. Because the energy
associated with the "masked LPS" is lower than that associated with
"active LPS", the LPS remains stabilized in this masked form. The
inventive methods described herein effectively destabilize this
masked LPS, thus raising its energy to a level above that of masked
LPS, from where LPS can again fall back down in energy into
aggregated form (far right of diagram). It as assumed that the
reconfiguring modulator plays a key role in mediating this rescue
of LPS from solubilized (masked) to aggregate (unmasked) form.
GENERAL
It is to be understood that the foregoing general description as
well as the following detailed description are exemplary and
explanatory only and are not restrictive of the invention as
claimed. In this application, the use of the singular includes the
plural unless specifically stated otherwise. In this application,
the use of "or" means "and/or" unless stated otherwise. Further,
the use of the term "including" as well as other grammatical forms
such as "includes" and "included", is not limiting. In the same
sense, the use of the term "comprising" as well as other
grammatical forms such as "comprises" and "comprised" is not
limiting. Section headings throughout the description are for
organizational purposes only. They are in particular not intended
as limiting for the various embodiments described therein, and it
is to be understood that elements and embodiments described under
one subheading may be freely combined with elements and embodiments
described under another subheading.
In the foregoing, subsequent description the claims, the features
of any one embodiment are intended as being combinable with those
of any other embodiment. Such combinations of one or more features
in any one embodiment with one or more features in any other
embodiment belong to the disclosure of the present application as
filed.
All documents or portions of documents cited in this application,
including but not limited to patents, patent applications,
articles, monographs, books, treaties and regulations, are hereby
expressly incorporated by reference in their entirety for any
purpose.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to an aqueous composition comprising
a protein and an aliphatic compound with C.sub.8-C.sub.16 as the
main chain and which compound preferably has a substitution by one
or more heteroatoms.
The aqueous composition may preferably be a pharmaceutical
composition containing a protein to which the aliphatic compound is
added. The addition of the aliphatic compound helps to improve the
detectability of a potential contamination of the composition by an
LPS. As stated in other parts of this application, LPS might escape
detection by conventional endotoxin tests because of being masked
by some constituents of protein-containing compositions.
According to a preferred embodiment, the aliphatic compound is a
branched compound with at least one substitution in the main chain
wherein the substitution may be selected from methyl, ethyl, propyl
and butyl groups.
The main chain of the aliphatic compound is as defined elsewhere
herein.
According to a further preferred embodiment, the main chain is
selected from a C.sub.8-C.sub.16 alkyl, C.sub.8-C.sub.16 alkenyl
and C.sub.8-C.sub.16 alkynyl. The main chain may contain one or
more double bonds and/or one or more triple bonds, whereas a
saturated alkyl chain is the more preferred embodiment.
According to a further preferred embodiment, the heteroatom that
may form part of the aliphatic compound is selected from O, S and
N, whereas O is the more preferred substitution.
A further preferred aliphatic compound is selected from an alkanol,
which is preferably an unbranched alkanol, more preferably a
1-alkanol and most preferably 1-dodecanol.
The aliphatic compound is assumed to stabilize a potentially
contaminating LPS molecule in a form that renders LPS more
susceptible to detection by conventional endotoxin test kits such
as the EndoLISA.RTM. by Hyglos GmbH.
Compositions that might be rendered more susceptible to the
detection of endotoxin often contain detergents which may be
selected from an anionic detergent, a cationic detergent, a
nonionic detergent, an amphoteric detergent and any combination
thereof. Preferred detergents that may be used in such compositions
may be selected from: an anionic detergent which can be chosen from
the group consisting of: alkyl sulfates, preferably ammonium lauryl
sulfate or sodium lauryl sulfate (SDS); alkyl-ether sulfates,
preferably sodium laureth sulfate or sodium myreth sulfate;
cholesterol sulfate; sulfonates, preferably dodecylbenzensulfonate,
sodiumlauryl sulfoacetate or xylene sulfonate; alkyl sulfo
succinates, preferably disodium lauryl sulfosuccinate; sulfoxides,
preferably dodecyl methyl sulfoxide; phosphates, preferably
trilaureth-4 phosphate; and carboxylates, preferably sodium
stearate or sodium lauroyl sarcosinate;
a cationic detergent which can be chosen from the group consisting
of: primary amines; secondary amines; tertiary amines; and
quaternary ammonium cations such as alkyltrimethylammonium salts
(preferably cetyl trimethylammonium bromide (CTAB); or cetyl
trimethylammonium chloride (CTAC)); cetylpyridinium chloride (CPC);
quaternary ammonium detergents, preferably
tris[2-(2-hydroxyethoxy)ethyl]-octadecyl-ammonium phosphate
(Quaternium 52); and hydroxyethylcellulose ethoxylate, quaternized
(Polyquaternium-10);
a nonionic detergent which can be chosen from the group consisting
of: polyoxyethylene glycol sorbitan alkyl esters (polysorbates),
preferably polysorbate 20 (TWEEN.TM.-20), polysorbate 40,
polysorbate 60 or polysorbate 80 (TWEEN.TM.-80); polyoxyethylene
glycol alkyl ethers; polyoxypropylene glycol alkyl ethers;
glucoside alkyl ethers; polyoxyethylene glycol octylphenol ethers;
polyoxyethylene glycol alkylphenol ethers; glycerol alkyl esters;
sorbitan alkyl esters; block copolymers of polyethylene glycol and
polypropylene glycol; cocamide MEA; sterols, preferably
cholesterol; cyclodextrins; poloxamers, preferably Pluronic block
polymers; and cocamide DEA;
an amphoteric detergent which can be chosen from the group
consisting of: CHAPS
(3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate);
sultaines, preferably cocamidopropyl hydroxysultaine; betaines,
preferably cocamidopropyl betaine; amino oxides, preferably
palmitamine oxide, laurylamine oxide and amine oxide of general
formula R.sup.3N.sup.+O.sup.-, wherein R.sup.3 is C.sub.8-C.sub.18
alkyl, C.sub.8-C.sub.18 alkenyl, or C.sub.8-C.sub.18 alkynyl; and
lecithin.
According to a further preferred embodiment, the detergent is
selected from a polysorbate, preferably Polysorbate 20 and
Polysorbate 80, a poloxamer, preferably Poloxamer 188, an
octoxynol, preferably an Octoxynol 9, an alkylamine oxide,
preferably laurylamine oxide, a quaternary ammonium salt,
preferably tris[2-(2-hydroxyethoxy)ethyl]-octadecyl-ammonium
phosphate, an alkyl phosphate, preferably trilaureth-4 phosphate,
and a stearate, preferably sodium stearate.
In a preferred aqueous composition, the protein is chosen from an
antibody, an antibody fragment, a hormone, an enzyme, a fusion
protein, a protein conjugate and any combination thereof, which
proteins are frequently used as the active agent of pharmaceutical
preparations where specific care must be taken that LPS does not
remain undetected in the quality control of pharmaceuticals.
In a further preferred embodiment, the antibody fragment is
selected from a Fab, a Fab', a F(ab')2 and an Fv, a single chain
antibody and any combination thereof.
In a further preferred embodiment, the aqueous composition, in
addition to the active pharmaceutical ingredient, which may be the
protein mentioned above, may contain an additional protein selected
from an albumin, which is preferably human serum albumin, bovine
serum albumin and/or ovalbumin. The further protein may be of
assistance in rendering a potential LPS contamination more
detectable by conventional endotoxin tests such as the ones
mentioned above.
In a further preferred embodiment, the aqueous composition may
comprise a chaotropic agent, a cation or a combination thereof. The
same ingredients also can help to bring a potential LPS
contamination into a form that is more susceptible to detection by
an endotoxin test by Hyglos GmbH.
According to a further preferred embodiment, the chaotropic agent
is selected from urea, guanidinium chloride, butanol, ethanol,
lithium perchlorate, lithium acetate, magnesium chloride, phenol,
propanol and thiourea.
According to a further preferred embodiment, the cation is a
divalent cation, preferably selected from Ca2+, Mg2+, Sr2+ and
Zn2+.
According to a further preferred embodiment, the further protein,
which may be an albumin, is present in a concentration in the range
from 0.1-20 mg/ml, preferably in the range from 1-10 mg/ml, more
preferably in an amount of 10 mg/ml.
In a further preferred embodiment, the aliphatic compound is
present in the concentration from 0.01-100 mM, preferably in a
concentration from 0.1-10 mM. This concentration range is in
particular preferred for an 1-alkanol, preferably 1-dodecanol.
In a further preferred embodiment, the detergent is present in a
concentration from 0.001-1.0 wt %, preferably 0.05-0.5 wt %,
preferably from 0.02-0.2 wt %.
In a further preferred embodiment, the chaotopic agent is present
in a concentration from 1 mM-1 M, preferably from 25-200 mM,
preferably from 10 mM-100 mM.
In a further preferred embodiment, the divalent cation is present
in a concentration from 1-400 mM, preferably in a concentration
from 10-200 mM, more preferably in a concentration from 50-100
mM.
In a further preferred embodiment, the pH of the composition is in
the range from 2-12, preferably in the range from pH 5-10.
In a further preferred embodiment, the composition contains Factor
C protein, which is a component used in for conventional endotoxin
assays.
In a preferred embodiment, the Factor C protein is a recombinant
Factor C protein.
A very preferred aqueous composition comprises a protein,
preferably an antibody, in combination with a 1-alkanol, preferably
1-dodecanol in a concentration range from 0.1-10 mM, a detergent of
claim 8 in a concentration range from 0.002-0.2 wt %, a divalent
cation, preferably Ca2+, in a concentration range from 10-200 mM,
and a pH from 5 to 10.
A further very preferred aqueous composition is as set out above in
the immediately preceding paragraph, and further comprising a
chaotropic agent, preferably guanidinium chloride, in the
concentration range from 10 mM-100 mM.
In the above compositions, LPS, if present, will be susceptible to
detection by conventional endotoxin assays such as the EndoLisa of
Hyglos GmbH.
As mentioned above, one disclosure relates to a method of unmasking
an endotoxin in a composition, preferably a pharmaceutical
composition, comprising an endotoxin masker and suspected of
comprising said endotoxin, said method comprising the step of
adding to said composition a modulator capable of unmasking said
endotoxin, e.g. by releasing said endotoxin, if present, from a
complex between said endotoxin and said endotoxin masker. The
pharmaceutical composition will in most cases be an aqueous
composition.
A further disclosure relates to a method of detecting an endotoxin
in a composition, preferably a pharmaceutical composition,
comprising an endotoxin masker and suspected of comprising said
endotoxin, said method comprising the steps of: adding to said
composition a modulator capable of unmasking said endotoxin, e.g.
by releasing said endotoxin, if present, from a complex between
said endotoxin and said endotoxin masker; and detecting said
endotoxin by means of a detection method. The pharmaceutical
composition will in most cases be an aqueous composition.
Endotoxin
The term "endotoxin" refers to a molecule produced on the surface
of bacteria in particular gram-negative bacteria, that is bacteria
which, because of their thin peptidoglycan layer sandwiched between
an inner cell membrane and a bacterial outer membrane, do not
retain the crystal violet stain used in the Gram staining method of
bacterial differentiation and therefore evade positive detection by
this method. Specifically, endotoxins are biologically active
substances present in the outer membrane of gram-negative bacteria.
One common class of endotoxins is lipopolysaccharides (LPS). For
the purposes of the present application, the terms "endotoxin" and
"LPS" are used interchangeably. As is discussed elsewhere herein,
however, it is understood that there exist different types of LPS,
e.g. derived from different sources, and that the terms "endotoxin"
and "LPS" are intended to encompass these different types of LPS.
Endotoxins are located on the surface of bacteria and, together
with proteins and phospholipids, form the outer bacterial membrane.
Generally, LPS is made up of two parts with different chemical and
physical properties; a hydrophilic sugar domain (the
polysaccharide) and a hydrophobic lipid domain (lipid A). Two
distinct regions can be recognized in the polysaccharide: the core
oligosaccharide and the O-specific polysaccharide (M. A.
Freudenberg, C. Galanos, Bacterial Lipopolysaccharides: Structure,
Metabolism and Mechanisms of Action, Intern. Rev. Immunol. 6,
1990).
The lipid A is highly hydrophobic and is the endotoxically active
part of the molecule. Lipid A is typically composed of a
beta-D-GlcN-(1-6)-alpha-D-GlcN disaccharide carrying two phosphoryl
groups. Up to four acyl chains are attached to this structure.
These chains can then in turn be substituted by further fatty
acids, which can vary quite considerably between species in their
nature, number, length, order and saturation. Covalently attached
to the lipid A is the core section of the molecule which can itself
be formally divided into inner and outer core. The inner core is
proximal to the lipid A and contains unusual sugars like
3-deoxy-D-manno-octulosonic acid (KDO). The outer core extends
further from the bacterial surface and is more likely to consist of
more common sugars such as hexoses and hexosamines. Onto this is
attached, in most cases, a polymer of repeating saccharide subunits
called the O-polysaccharide, also typically composed of common
sugars. This O-polysaccharide is not ubiquitous, however, as it is
seen to be truncated or lacking in a number of Gram-negative
strains. In addition, certain strains carry mutations in the
otherwise well-conserved locus and are termed "rough mutants" to
differentiate them from the wild-type "smooth" strains which
express O-polysaccharide bearing LPS (C. Erridge, E.
Bennett-Guerrero, I. Poxton, Structure and function of
lipopolysaccharides, Microbes and Infection, 2002). Copious
information relating to endotoxins, e.g. LPS, as well as their
impact on health may be found in the book "Endotoxin in Health and
Disease", edited by Helmut Brade, Steven M. Opal, Stefanie N. Vogel
and David C. Morrison, 1999, published by Marcel Dekker, Inc., ISBN
0-8247-1944-1.
As mentioned above, endotoxin may derive from different bacterial
sources. The chemical nature of endotoxin may vary slightly from
source to source. For instance, endotoxins derived from different
bacterial sources may differ slightly in the length of the
aliphatic chains in the aliphatic amides and aliphatic acid esters
of the lipid A domain. However, despite slight variations in
endotoxin structure from source to source, the same basic structure
as described herein above applies for most if not all endotoxins,
implying a similar mode of action, and a correspondingly similar
mode of influencing endotoxin behavior regardless of the bacterial
species of origin. Examples of known endotoxins include those
derived from e.g. E. coli, e.g. E. coli O55:B5 (such as available
from Sigma as product number L2637-5MG) or E. coli K 12; S. abortus
equi (such as available from Acila as product number 1220302);
Klebsiella pneumonia; Morganella morganii; Yersinia enterocolitica;
Serratia marcescens; Neisseria, e.g. Neisseria meningitis;
Acinetobacter baumanni; Enterobacter cloacae, e.g. naturally
occurring endotoxin (NOE); Pseudomonas, e.g. Pseudomonas
aeruginosa; Salmonella, e.g. Salmonella enteric; Shigella;
Haemophilus influenza; Bordatella pertussis; and Vibrio cholerae.
It is to be understood that this list is merely exemplary and in no
way restricts the term "endotoxin" as used herein.
Endotoxin Masker
The term "endotoxin masker" refers to a substance which, in
solution with the endotoxin, renders the endotoxin undetectable by
available detection methods, e.g. by limulus amebocyte lysate (LAL)
tests. Typically, endotoxin is detectable when it exists in
solution in aggregated form, i.e. in a form in which multiple, or
least two endotoxin moieties are held together in spatial proximity
by non-covalent interactions such as electrostatic interactions,
hydrophobic interactions, Van der Waals interactions or any
combination thereof. However, endotoxin becomes significantly less
active (undetectable) as measured by common detection systems, i.e.
is masked, when its active aggregation state is changed such that
the endotoxin becomes solubilized as individual molecules of
endotoxin. It can be assumed that discrete molecular entities of
endotoxin are stabilized, for example, by detergents present in the
solution. Such detergents are assumed to stabilize individual
endotoxin moieties by forming detergent micelles in which the
individual endotoxin moieties become embedded and are no longer
capable of reacting with Factor C in commercially available
endotoxin assays. Certain proteins may also effect or contribute to
stabilization of endotoxin in undetectable soluble form. For
instance, such proteins may present the endotoxin with binding
clefts offering individual endotoxin molecules a suitable
environment for stable binding, thereby breaking up otherwise
detectable endotoxin aggregates and/or preventing the endotoxin
molecules from interacting with Factor C in available endotoxin
assays.
It is assumed that at least two molecules of endotoxin, that is at
least two molecules of LPS, must form an aggregate in order to be
detectable by commercially available endotoxin tests such as the
EndoLISA.RTM. test kit available from Hyglos GmbH and LAL-based
tests.
In fact, several publications show that endotoxin aggregates are
significantly more biologically active than disaggregated
endotoxins (M. Mueller, B. Lindner, S. Kusumoto, K. Fukase, A, B.
Schromm, U. Seydel, Aggregates are the biologically acitve units of
endotoxin, The Journal of biological Chemistry, 2004; A. Shnyra, K.
Hultenby, A. Lindberg, Role of the physical state of Salmonella
Lipopolysaccharide in expression of biological and endotoxic
properties, Infection and Immunity, 1993). Furthermore, the
activation of Factor C, described by Tan et al. (N. S. Tan, M. L.
P. NG, Y. H Yau, P. K. W. Chong, B Ho, J. L. Ding, Definition of
endotoxin binding sites in horseshoe crab Factor C recombinant
sushi proteins and neutralization of endotoxin by sushi peptides,
The FASEB Journal, 2000), is indicated as a cooperative binding
mechanism. Here, as mentioned above, at least two LPS molecules are
required for activation of Factor C, which is the key factor in
limulus based detection methods such as the EndoLISA kit available
from Hyglos GmbH.
Examples of endotoxin maskers which are detergents include anionic
detergents, cationic detergents, nonionic detergents and amphoteric
detergents, and any combination thereof.
Examples of anionic detergents which may function as detergent
endotoxin maskers in the sense of the invention include alkyl
sulfates such as for example ammonium lauryl sulfate or sodium
lauryl sulfate (SDS); alkyl-ether sulfates such as for example
sodium laureth sulfate or sodium myreth sulfate; cholesterol
sulfate; sulfonates such as for example dodecylbenzensulfonate,
sodiumlauryl sulfoacetate or xylene sulfonate; alkyl sulfo
succinates such as for example disodium lauryl sulfosuccinate;
sulfoxides such as for example dodecyl methyl sulfoxide; phosphates
such as for example trilaureth-4 phosphate; and carboxylates such
as for example sodium stearate or sodium lauroyl sarcosinate.
Examples of cationic detergents which may function as endotoxin
maskers in the sense of the invention include primary amines;
secondary amines; tertiary amines; and quaternary ammonium cations
such as for example alkyltrimethylammonium salts (e.g. cetyl
trimethylammonium bromide (CTAB) or cetyl trimethylammonium
chloride (CTAC)); cetylpyridinium chloride (CPC); quaternary
ammonium detergents such as for example
tris[2-(2-hydroxyethoxy)ethyl]-octadecyl-ammonium phosphate
(Quaternium 52); and hydroxyethylcellulose ethoxylate, quaternized
(Polyquaternium-10).
Nonionic detergents which may function as detergent endotoxin
maskers in the sense of the invention include polyoxyethylene
glycol sorbitan alkyl esters (polysorbates) such as for example
polysorbate 20 (TWEEN.TM.-20), polysorbate 40, polysorbate 60 or
polysorbate 80 (TWEEN.TM.-80); polyoxyethylene glycol alkyl ethers;
polyoxypropylene glycol alkyl ethers; glucoside alkyl ethers;
polyoxyethylene glycol octylphenol ethers; polyoxyethylene glycol
alkylphenol ethers; glycerol alkyl esters; sorbitan alkyl esters;
block copolymers of polyethylene glycol and polypropylene glycol;
cocamide MEA; sterols such as for example cholesterol;
cyclodextrans; poloxamers such as for example Pluronic block
polymers (for example
HO--(CH.sub.2CH.sub.2O).sub.n/2--(CH.sub.2CH(CH.sub.3)O).sub.m--(CH.sub.2-
CH.sub.2O).sub.n/2--H, with n=200 and m=65 for F127 and n=4.5 and
m=31 for F61) and cocamide DEA.
Amphoteric detergents which may function as detergent endotoxin
maskers in the sense of the invention include CHAPS
(3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate);
sultaines, such as for example cocamidopropyl hydroxysultaine;
betaines, such as for example cocamidopropyl betaine; amine oxides
such as for example palmitamine oxide, laurylamine oxide and amine
oxide of general formula R.sup.3N.sup.+O.sup.-, wherein R.sup.3 is
C.sub.8-C.sub.18 alkyl, C.sub.8-C.sub.18 alkenyl or
C.sub.8-C.sub.18 alkynyl; and lecithin. Specifically, R.sup.3 in
the above general formula R.sup.3N.sup.+O.sup.- may be any of
C.sub.8 alkyl, C.sub.9 alkyl, C.sub.10 alkyl, C.sub.11 alkyl,
C.sub.12 alkyl, C.sub.13 alkyl, C.sub.14 alkyl, C.sub.15 alkyl,
C.sub.16 alkyl, C.sub.17 alkyl or C.sub.18 alkyl; or C.sub.8
alkenyl, C.sub.9 alkenyl, Co alkenyl, C.sub.11 alkenyl, C.sub.12
alkenyl, C.sub.13 alkenyl, C.sub.14 alkenyl, C.sub.15 alkenyl,
C.sub.16 alkenyl, C.sub.17 alkenyl or C.sub.18 alkenyl; or C.sub.8
alkynyl, C.sub.9 alkynyl, C.sub.10 alkynyl, C.sub.11 alkynyl,
C.sub.12 alkynyl, C.sub.13 alkynyl, C.sub.14 alkynyl, C.sub.15
alkynyl, C.sub.16 alkynyl, C.sub.17 alkynyl or C.sub.18
alkynyl.
Alternatively or in addition to any of the endotoxin maskers
indicated above (alone or in combination), the endotoxin masker may
also be an active pharmaceutical ingredient (API). This API may
exist in solution together with or without any of the detergent
endotoxin maskers indicated above. If the API exists together with
a detergent endotoxin masker in solution, the masking effect may be
more pronounced, and more stringent measures may be necessary to
liberate masked endotoxin from the endotoxin masker, as is
discussed in greater detail below. APIs which may especially
engender or augment the masking of endotoxin present in the
solution are protein APIs, for example an antibody; an antibody
fragment; a hormone; an enzyme; a fusion protein; a protein
conjugate; and any combination thereof. When the protein API is an
antibody fragment, the antibody fragment may be preferably chosen
from the group consisting of: Fab; a Fab'; a F(ab')2; an Fv; a
single chain antibody; and any combination thereof. When the
protein API is an antibody, the antibody may be preferably chosen
from the group consisting of: a fully human antibody; an
anti-idiotype antibody; a humanized antibody; a bispecific
antibody; a chimeric antibody; a CDR-grafted antibody; a monoclonal
antibody; a polyclonal antibody; and any combination thereof.
Alternatively or in addition to the above, the API may also be a
small organic molecule. The skilled person understands what is
meant by the term "small organic molecule" or "small molecule".
This is a molecule with a molecular weight of no more than 300
g/mol, 400 g/mol, 500 g/mol, 600 g/mol, 700 g/mol, 800 g/mol, 900
g/mol or, preferably, 1000 g/mol.
Generally, an endotoxin masker, whether a detergent or a protein,
will have the characteristic of shifting the equilibrium between
solubilized and aggregated endotoxin in the direction of
solubilized endotoxin which is not detectable by available
endotoxin assays. It is this shifting of endotoxin into an
undetectable form which is referred to as "masking" herein. As
mentioned above, the form in which the endotoxin is solubilized may
for example include endotoxin a) being embedded in the lipid layer
of a micelle formed by a detergent; b) being bound on or in a
protein, e.g. in a suitable binding cleft of appropriate steric and
electrostatic environment formed on the surface of an active
pharmaceutical agent, e.g. a protein; or c) participating in a
combination of these two possibilities. Regardless of the form in
which endotoxin is solubilized so as to energetically disfavor the
aggregate form, however, the net effect is that individual
molecules of endotoxin which would otherwise be aggregated and
therefore detectable, are individually stabilized and, in this
individualized (solubilized) form, become and remain undetectable,
i.e. masked.
Although undetectable, however, such stabilized endotoxin molecules
in solution can nevertheless engender and/or contribute to the
sorts of pyrogenic and/or toxic reactions outlined above when
administered to subjects. This danger is especially acute in
pharmaceutical formulations, since pharmaceutical formulations
often contain a detergent to solubilize an API, e.g. a protein API,
which, without the detergent, would be insoluble at the
concentrations provided in the pharmaceutical formulation. In
rendering the API, e.g. protein API, soluble by including
detergent, then, one often unwittingly destroys the very
aggregation of endotoxin which is needed for detection of this
endotoxin. Thus, when the endotoxin masker is a detergent, the very
measure employed to formulate the API, e.g. protein API, in
acceptable form and concentration also has the potential to mask
endotoxin in solution.
As mentioned above the endotoxin masker may also be a protein, for
instance the API itself. This scenario may arise in conjunction
with the presence of a detergent endotoxin masker or, in the event
that no detergent is present, may also arise in the absence of a
detergent endotoxin masker. In this latter case, the API, in
particular a protein API, may offer the endotoxin a sufficient
environment for stable binding on or in such protein such that the
endotoxin is masked by the API alone, i.e. without any detergent
being necessary to mask endotoxin, rendering it undetectable. In
the event that the endotoxin masker is a protein, this protein may
be the API itself, or may alternatively or additionally be a
protein in solution which is different from the API. Generally, any
protein having an appropriate steric and electrostatic environment
to stabilize individual molecules of endotoxin, for instance
individual molecules of LPS could potentially effect or contribute
to the masking of endotoxin.
It is a hallmark of the invention that when the endotoxin masker is
a protein, either alone or together with an additional endotoxin
masker such as e.g. a detergent endotoxin masker, unmasking the
endotoxin leaves the protein endotoxin masker chemically unaltered
following unmasking. In particular, unmasking the endotoxin does
not cleave or otherwise degrade the protein endotoxin masker (e.g.
protein API).
In scenarios of the type described above, individual molecules of
endotoxin which would otherwise remain in aggregates and therefore
detectable, are stabilized at one or more such surface locations on
or in said protein. As is the case for detergent micelles, such
stabilization shifts the equilibrium existing between solubilized
(undetectable) and aggregated (detectable) in the direction of
solubilized (undetectable) endotoxin. As mentioned above, one may
imagine such a shift of equilibrium toward the solubilized
(undetectable) form as being especially pronounced in the event
that a solution comprises both detergent and one or more proteins
with the above characteristics, since in such cases the
stabilization of individual molecules of endotoxin out of its
aggregate form by the endotoxin masker may ensue both in the form
of stabilization in micelles as well as on the surface of proteins.
In such scenarios, more stringent measures may be required to shift
said equilibrium toward the aggregate endotoxin form which is then
detectable. These are discussed in more detail in the context of
illustrative scenarios further below (FIGS. 1-6).
Modulator
The term "modulator" as used herein refers to one or more compounds
which, alone or in concert, render(s) a masked endotoxin
susceptible to detection by an endotoxin assay (such as the
EndoLISA.RTM. detection assay available from Hyglos GmbH). The term
"modulator" as used herein may encompass both single as well as
multiple components which achieve this end. In some instances
herein below, reference is made to a "modulator system", although
the term "modulator" is sometimes used to designate multiple
modulator substances which are intended to work in concert. This
refers to a multi-component modulator comprising multiple
substances which act in concert to render a masked endotoxin
detectable by an endotoxin assay. The different components of a
modulator system may be incorporated for different reasons, i.e. to
take advantage of different functions of modulator substances which
affect the stability of a complex between endotoxin and endotoxin
masker in different ways. For ease of reference, one may for
example refer to different kinds of modulator which may be employed
alone or together to unmask endotoxin: "Disrupting modulator": A
"disrupting modulator" is a modulator which completely or partially
breaks up a complex between an endotoxin masker and an endotoxin.
When the endotoxin masker is a detergent, and the endotoxin is
masked in solubilized form inserted in the lipid layer of a masking
detergent micelle, then a modulator which disrupts such a detergent
micelle so as to liberate the endotoxin would be referred to as a
disrupting modulator. As discussed in greater detail below,
1-dodecanol is one such disrupting modulator. A disrupting
modulator, for example 1-dodecanol, 1-decanoic acid or sodium octyl
sulfate (SOS) may advantageously be used in a concentration range
of 0.01-100 mM, preferably in a concentration range of 0.1-10 mM,
preferably at a concentration of 10 mM in the unmasking process. In
some cases, the disrupting modulator may also simultaneously
function as a reconfiguring modulator, described below. "Adsorbing
modulator": An "adsorbing modulator" is a modulator which has the
ability to bind substances which would otherwise stabilize the
endotoxin in solubilized and therefore non-detectable form. For
instance, when the endotoxin masker is a detergent as e.g.
contained in some pharmaceutical compositions, then a modulator
which binds molecules of the detergent and in this way contributes
to the breakdown of endotoxin-stabilizing micelles would be
referred to as an adsorbing modulator. As discussed in greater
detail below, BSA is one such adsorbing modulator. An adsorbing
modulator, for example BSA may advantageously be used in a
concentration range of 0.1-20 mg/mL, preferably in a concentration
range of 1-10 mg/mL, preferably at a concentration of 10 mg/ml in
the unmasking process. "Displacing modulator": A "displacing
modulator" is a modulator which has the ability to completely or
partially displace a molecule of endotoxin from its stable binding
position in or on an endotoxin masker. For instance, when the
endotoxin masker is a protein, and the endotoxin is bound in or on
a protein which stabilizes the endotoxin in undetectable form, then
a modulator which has the ability to replace the endotoxin in or on
the protein, e.g. by means of hydrophobic interactions, would be
referred to as a displacing modulator. As discussed in greater
detail below, SDS is one such displacing modulator. A displacing
modulator, for example SDS, may advantageously be used in a
concentration range of 0.01-1%, preferably in a concentration range
of 0.05-0.5%, preferably at a concentration of 0.1% in the
unmasking process. "Reconfiguring modulator": A "reconfiguring
modulator" is a modulator which has the ability to transiently
stabilize endotoxin following its liberation from the endotoxin
masker (e.g. by a disrupting modulator or displacing modulator, as
discussed above), thus helping the liberated, solubilized
(undetectable) endotoxin to adopt an aggregated (detectable) form.
With the help of the reconfiguring modulator, solubilized endotoxin
becomes reconfigured as aggregated endotoxin. As discussed in
greater detail below, 1-dodecanol is one such reconfiguring
modulator. A reconfiguring modulator, for example 1-dodecanol,
1-decanoic acid or sodium octyl sulfate (SOS) may advantageously be
used in a concentration range of 0.01-100 mM, preferably in a
concentration range of 0.1-10 mM in the unmasking process. In some
cases, the reconfiguring modulator may also simultaneously function
as a disrupting modulator, described above.
As will become clear herein below, the above types of modulator are
not mutually exclusive; that is, it is possible for a given
substance to have functionality as different kinds of modulators
above. One example is 1-dodecanol, which may be classified as both
a disrupting modulator (breaking up a detergent micelle) as well as
a reconfiguring modulator (transiently stabilizing the
micelle-liberated endotoxin so it can aggregate and become
detectable). Similarly SDS may be classified as a disrupting
modulator (breaking up existing micelles of another, non-SDS
detergent) and a displacing modulator (liberating endotoxin from
binding sites in or on any masking protein which may be present).
The classification as to the type of modulator depends on the
function that a substance in question plays in a particular
composition. However, since it is assumed that reconfiguring of the
endotoxin from solubilized into aggregated form will generally be
required in order to render the endotoxin detectable, the modulator
will normally comprise at least one component qualifying as a
"reconfiguring modulator".
As a further example, a substance which functions as a "displacing
modulator" when the endotoxin masker is a protein may in some cases
function as a "disrupting modulator" when the endotoxin masker is a
detergent. SDS is one example of such a substance, the
classification of which as to the type of modulator component
depends on the prevailing conditions. For instance, when the
endotoxin masker is a protein, SDS will generally function as a
displacing modulator, since it helps to displace the endotoxin
bound in or on the masking protein. However, when the endotoxin
masker is a detergent, then SDS, alone or together with another
modulator component, may function more as a disrupting modulator,
since in this case it promotes the liberation of endotoxin from the
lipid layer of detergent micelles by disrupting the micelles.
A modulator may contain one or more substances within the above
classifications. For instance, a single component modulator may
comprise only a disrupting modulator such as 1-dodecanol. A
dual-component modulator may comprise a mixture of a disrupting
modulator such as 1-dodecanol (also possibly functioning as a
reconfiguring modulator) and, depending on the nature of the
masking complex between endotoxin and endotoxin masker, one of an
adsorbing modulator such as BSA or a displacing modulator such as
SDS. A multi-component modulator may comprise a mixture of a
disrupting modulator such as 1-dodecanol (also possibly functioning
as a reconfiguring modulator) and, depending on the nature of the
masking complex between endotoxin and endotoxin masker, one each of
an adsorbing modulator such as BSA and a displacing modulator such
as SDS. As will be discussed in detail below, the complexity of the
modulator system chosen will depend on the nature of the complex
between endotoxin and endotoxin masker, and the surrounding
solution conditions which contribute to the stability of that
complex. From the above, it is clear that each new composition to
be analyzed for the presence of endotoxin may require its own
customized modulator composition in order to render the masked
endotoxin susceptible to detection. The identification of a
suitable modulator for a given composition or formulation to be
tested can however be accomplished by routine experimentation, as
will be shown further below.
As mentioned above, in its most general sense, the modulator is
assumed to destabilize a complex between endotoxin and an endotoxin
masker and to promote the liberation of the endotoxin from the
endotoxin masker. In this way, the modulator or modulator system
effectively shifts the equilibrium from a solubilized
(undetectable) state toward an aggregated (detectable) state.
The present inventors have surprisingly found that endotoxin which
is present but undetectable in solution remains undetectable
because, as assumed, the endotoxin remains stably solubilized in
detergent micelles and/or bound to surface structures of proteins
present in the solution. Individually stabilized in this form, the
endotoxin molecules evade detection. However, the present inventors
have found that solution conditions can be manipulated such that
solubilized endotoxin is rendered into a form which can be
detected. In some instances, multiple manipulations of solution
conditions may be required to reach this end and the stringency of
the measure or measures taken to effect the desired shift in
equilibrium toward an aggregated state will vary depending on the
degree to which the endotoxin masker stabilizes the endotoxin in
solubilized form, as mentioned above. But generally, the
manipulations performed in accordance with the invention as
described herein should be understood in the context of the overall
aim of shifting the equilibrium of endotoxin from a solubilized
state to an aggregated state such that it can be detected.
In order to accomplish the above, the "modulator" will generally
include an amphiphilic molecule which competes for binding between
the lipid component of endotoxin and the endotoxin masker, thus
weakening the interaction between the former and the latter. Such
competitive binding will generally be accomplished by providing at
least one component of the modulator system in a form which is
structurally similar to the (amphiphilic) lipid component of the
endotoxin such that the former may displace the latter in its
stabilized interaction with the endotoxin masker.
For instance, in the event the endotoxin masker is a detergent, a
suitable disrupting modulator will generally include an amphiphilic
compound capable of stably inserting i.e. between the amphiphilic
detergent molecules and the similarly amphiphilic lipid portion of
the endotoxin. When the endotoxin masker is a detergent, an
amphiphilic disrupting modulator will therefore elicit several
effects in parallel which are conducive to an overall shift in
equilibrium from a solubilized toward an aggregated form of
endotoxin. First, providing a modulator system comprising at least
one amphiphilic disrupting modulator disrupts the lipophilic
interactions underlying the detergent micelles such that these
micelles are broken up. Since endotoxin was previously solubilized
(and therefore masked) by insertion of its lipid component into the
lipid layer of the detergent micelles, the breakup of the micelles
removes this stabilizing force and results in the liberation of
previously embedded endotoxin. The role of the disrupting modulator
in the event that the endotoxin masker is or includes a detergent
is thus to break up detergent micelles.
Further, the amphiphilic character of the disrupting modulator may
also enable it to associate with the lipid component of the
endotoxin, once the endotoxin is liberated from its detergent
micelles as described above. This interaction between the
amphiphilic disrupting modulator and the lipid component of the
endotoxin has the effect of chaperoning the endotoxin in aqueous
solution following its liberation from the stabilizing detergent
micelles. In this event, the disrupting modulator would have a
double function as a reconfiguring modulator. When the disrupting
modulator is amphiphilic in character, it is not excluded that it
may be capable for forming micelles of its own. However, the
unmasking effect will generally be greatest when the amphiphilic
disrupting modulator does not form micelles of its own which might
simply swap one solubilized and therefore masked endotoxin state
for another. A key role of the reconfiguring modulator is thus to
transiently stabilize liberated endotoxin (albeit less than in its
previous complex with the endotoxin masker), effectively
chaperoning the endotoxin into an aggregated and therefore
detectable state.
Thus temporarily chaperoned in solution, the liberated endotoxin is
then free to aggregate into a form which is detectable and
therefore "unmasked". Whether or not further manipulation of
solution conditions beyond addition of the modulator or modulator
system is necessary to shift equilibrium towards this aggregated,
detectable form will generally depend on the conditions prevailing
in solution and the initial stability of the endotoxin as complexed
with the endotoxin masker.
In another scenario already contemplated above, the endotoxin
masker is not, or not only a detergent, but may also be or comprise
a protein with binding clefts on its surface suitable to stably
bind individual moieties of endotoxin such that it cannot be
detected. In this event, similar considerations pertaining to the
modulator apply as set out above. For instance, use of a disrupting
(amphiphilic) modulator and/or a displacing modulator in the event
that the endotoxin is or comprises a protein has the effect that
the modulator disrupts the lipophilic interactions existing between
lipophilic amino acid side chains of the protein (endotoxin masker)
and the lipid component of the endotoxin. Because the disrupting
modulator and/or displacing modulator is/are likely to be
amphiphilic in character, the modulator(s) would also be able to
disrupt electrostatic interactions existing between polar and/or
ionized side chains within the protein (endotoxin masker) and polar
groups within the core and/or O-antigen polysaccharide regions of
the endotoxin. With these stabilizing interactions disrupted, the
endotoxin which was previously masked by a protein endotoxin masker
is thus displaced from its previous complex with the protein, and
is chaperoned in solution into an aggregated state by association
with a reconfiguration modulator as described above.
As described above for the case in which the endotoxin masker is a
detergent in the absence of a protein endotoxin masker, the
liberated and reconfiguation modulator-chaperoned endotoxin is then
free to aggregate into a form which is detectable and therefore
"unmasked". Whether or not further manipulation of solution
conditions beyond addition of the components of the modulator
system is necessary to shift equilibrium towards this aggregated,
detectable endotoxin form will generally depend on the conditions
prevailing in solution and the initial stability of the endotoxin
as complexed with the endotoxin masker.
The modulator, e.g. the disrupting modulator, the displacing
modulator and/or the reconfiguring modulator may in certain
embodiments comprise a first heteroatom-substituted aliphatic,
wherein the main chain of the first heteroatom-substituted
aliphatic comprises 8 to 16 carbon atoms. As used herein, the term
"main chain" refers to the longest chain of the first
heteroatom-substituted aliphatic comprising 8 to 16 carbon atoms,
as numbered by standard IUPAC nomenclature. Specifically, the main
chain of the first heteroatom-substituted aliphatic may comprise 8,
9, 10, 11, 12, 13, 14, 15 or 16 carbon atoms. As used herein, the
term "heteroatom" refers to any atom other than carbon, to which a
carbon atom in the first heteroatom-substituted aliphatic is
covalently bound. Representative heteroatoms include oxygen,
nitrogen and sulfur atoms. In a further preferred embodiment, the
oxygen-substituted aliphatic is an aliphatic alcohol, in
particular, 1-dodecanol, that is the molecule given by the formula
HO--(CH.sub.2).sub.11--CH.sub.3. As mentioned above, 1-dodecanol is
especially well suited in many instances as a disrupting modulator
as well as, in most if not all instances, as a reconfiguring
modulator.
The reconfiguring modulator is assumed to play an especially
important, if not indispensible role in promoting an aggregated,
detectable form of endotoxin. The reconfiguring modulator may be a
heteroatom-substituted aliphatic comprising 8, 9, 10, 11, 12, 13,
14, 15 or 16 carbon atoms in its main chain. The term "main chain"
refers to the longest chain of the reconfiguring modulator, as
numbered by standard IUPAC nomenclature. As used herein, the term
"heteroatom" refers to any atom other than carbon, to which a
carbon atom in the first heteroatom-substituted aliphatic is
covalently bound. Representative heteroatoms include oxygen,
nitrogen and sulfur atoms. It is especially suitable when the
heteroatom is oxygen. Furthermore, the reconfiguring modulator may
be branched or unbranched, with the branched variants comprising
substitutions along the "main chain" as defined above. Said
substitutions may be e.g. methyl, ethyl, propyl and/or butyl. An
unbranched chain is preferred. The reconfiguring modulator may be
saturated to various extents, and may for example comprise a
C.sub.8 alkyl, C.sub.9 alkyl, C.sub.10 alkyl, C.sub.11 alkyl,
C.sub.12 alkyl, C.sub.13 alkyl, C.sub.14 alkyl, C.sub.15 alkyl or
C.sub.16 alkyl moiety; or a C.sub.8 alkenyl, C.sub.9 alkenyl,
C.sub.10 alkenyl, C.sub.11 alkenyl, C.sub.12 alkenyl, C.sub.13
alkenyl, C.sub.14 alkenyl, C.sub.15 alkenyl or C.sub.16 alkenyl
moiety; or a C.sub.8 alkynyl, C.sub.9 alkynyl, C.sub.10 alkynyl,
C.sub.11 alkynyl, C.sub.12 alkynyl, C.sub.13 alkynyl, C.sub.14
alkynyl, C.sub.15 alkynyl or C.sub.16 alkynyl moiety. Furthermore,
the reconfiguring modulator may contain any mixture of single,
double and triple carbon-carbon bonds. Especially suitable
reconfiguring modulators are saturated, i.e. comprise C.sub.8
alkyl, C.sub.9 alkyl, C.sub.10 alkyl, C.sub.11 alkyl, C.sub.12
alkyl, C.sub.13 alkyl, C.sub.14 alkyl, C.sub.15 alkyl or C.sub.16
alkyl. Especially suitable reconfiguring modulators comprise
C.sub.12 alkyl. Furthermore, the heteroatom may be of various
oxidation states. For instance, when the heteroatom is oxygen, the
oxygen may be in the form of an alcohol, an aldehyde or a
carboxylic acid.
Especially suitable as reconfiguring modulators are molecules in
unbranched alkanols, in particular unbranched 1-alkanols. Among
these, especially suitable are C.sub.12 alkanols, especially
1-dodecanol having the formula HO--(CH.sub.2).sub.11--CH.sub.3.
In further embodiments, the modulator system may include other
components in addition to said first heteroatom-substituted
aliphatic comprising 8 to 16 carbon atoms. For example, the
modulator system may additionally comprise a second
heteroatom-substituted aliphatic, e.g. as a disrupting modulator, a
displacing modulator and/or a reconfiguring modulator, wherein the
main chain of said second heteroatom-substituted aliphatic
comprises 8 to 16 carbon atoms. The "main chain" of the second
heteroatom-substituted aliphatic is defined as described above for
the first heteroatom-substituted aliphatic. Specifically, the main
chain of the second heteroatom-substituted aliphatic may comprise
8, 9, 10, 11, 12, 13, 14, 15 or 16 carbon atoms. The first
heteroatom-substituted aliphatic comprising 8 to 16 carbon atoms is
different than the second heteroatom-substituted aliphatic
comprising 8 to 16 carbon atoms. In a preferred embodiment, the
second heteroatom-substituted aliphatic which may be part of the
modulator is an oxygen-substituted aliphatic. In certain preferred
embodiments, this oxygen-substituted aliphatic is an aliphatic
sulfate, wherein it is especially preferred that this aliphatic
sulfate is sodium dodecyl sulfate (SDS). Thus, in a particularly
preferred embodiment of the invention, the modulator system
includes a first heteroatom-substituted aliphatic which is
1-dodecanol (e.g. as a disrupting modulator and/or a reconfiguring
modulator), and a second heteroatom-substituted aliphatic which is
SDS (as a further disrupting modulator and/or a displacing
modulator).
In a further embodiment, the modulator system as described above
may further comprise a protein capable of binding a detergent so as
to break up micelles formed by said detergent. Generally, the
detergent bound will be the endotoxin masker (when said endotoxin
masker is or comprises a detergent), and the principle by which the
protein capable of binding a detergent binds the detergent is
analogous to the principle described above, according to which a
protein which functions as an endotoxin masker sequesters portions
of the endotoxin molecule in or on its surface. In the present
embodiment, the protein capable of binding a detergent, when used
as part of the modulator, also bears on its surface areas of steric
and electrostatic compatibility with a portion or portions of
detergent molecules present in solution, which are sufficient to
bind or sequester detergent molecules, thus rendering them
unavailable for participation in micelles and thus breaking up any
detergent micelles which may be harbor endotoxin, or which may
serve to shift equilibrium away from an aggregated form of
endotoxin.
The inventors have found that albumin molecules are exceptionally
good at binding detergent. Thus, it is contemplated that in
addition to the first heteroatom-substituted aliphatic alone, or in
addition to the first heteroatom-substituted aliphatic in
combination with the second heteroatom-substituted aliphatic, the
modulator may additionally comprise a protein (adsorbing modulator)
capable of binding a detergent so as to break up micelles formed by
said detergent. In certain embodiments, the protein component of
the modulator may be an albumin, preferably human serum albumin
(HSA), bovine serum albumin (BSA) or ovalbumin (OVA).
It is additionally contemplated that the modulator may contain one
or more of each of the first heteroatom-substituted aliphatic
comprising 8 to 16 carbon atoms, the said second
heteroatom-substituted aliphatic comprising 8 to 16 carbon atoms
and said protein capable of binding a detergent so as to break up
micelles formed by said detergent. In a preferred embodiment of the
invention, the modulator comprises 1-dodecanol alone. In a further
preferred embodiment of the invention, the modulator comprises
1-dodecanol and SDS. In a further preferred embodiment of the
invention, the modulator comprises 1-dodecanol, SDS and HSA. In a
further preferred embodiment of the invention, the modulator
comprises 1-dodecanol, SDS and BSA.
Composition
As used herein, the term "composition" refers to a mixture
comprising (at least) an endotoxin masker. The endotoxin, even if
present and masked, remains undetectable in the composition. The
composition is preferably a pharmaceutical composition, e.g. a
composition comprising an active pharmaceutical ingredient, or API.
The term "composition" may be e.g. an extract; vaccine; any
composition suitable for parenteral administration, i.e.
parentalia; any composition suitable for intraperitoneal,
transdermal, subcutaneous or topical administration; a blood
product; a cell therapy solution, e.g. intact, living cells, for
example, T cells capable of fighting cancer cells; a gene therapy
solution, e.g. a solution capable of nucleic acid polymer delivery
into a patient's cells as a drug to treat disease; an implant or
medical device; or a composition resulting from rinsing or wiping
the surface of an object, said object for instance being a medical
device, an implant or a filling machine.
Detection Method
As used herein, the term "detection method" refers to a method
which is suitable for detecting endotoxin in solution. For example,
suitable methods in this regard are limulus based detection
methods, or is an enzyme linked immunosorbent assays (ELISA). The
limulus methods can be performed classically by using natural
derived lysate (J. Jorgensen, R. Smith, Perparation, Sensitivity,
and Specificity of Limulus Lysate for Endotoxin Assay, Applied
Microbiology, 1973) or recombinantly prepared Factor C (J. L. Ding,
B. Ho, A new era in pyrogen testing, Trends in Biotechnology,
2001). The most promising of such methods are enzyme-linked
affinitysorbent assays, using a solid phase for endotoxin capturing
and subsequent detection by recombinant version of a protein in the
LAL assay, Factor C. The EndoLISA.RTM. kit is one such
affinitysorbent assay (H. Grallert, S. Leopoldseder, M. Schuett, P.
Kurze, B. Buchberger, EndoLISA.RTM.: a novel and reliable method
for endotoxin detection, Nature Methods, 2011). The EndoLISA.RTM.
detection system is for example described in the book
"Pharmazeutische Mikrobiologie--Qualitatssicherung, Monitoring,
Betriebshygiene" by Michael Rieth, October 2012, Wiley-VCH,
Weinheim, ISBN 978-3-527-33087-4.
Agent which Influences Hydrogen Bonding Stability in Solution
According to a further embodiment of the invention, the above
methods of unmasking an endotoxin and/or the method of detecting an
endotoxin may further comprise the step of adding to said
composition an agent which influences hydrogen bonding stability in
solution. Generally, as used herein, an agent which influences
hydrogen bonding stability in solution modifies solution conditions
so as to destabilize the complex in which an individual molecule or
multiple molecules of endotoxin is/are solubilized and therefore
masked.
Not all complexes between endotoxin and endotoxin masker are the
same. In particular, the energy minima governing endotoxin
stabilization in certain masking complexes are different than those
governing endotoxin stabilization in other masking complexes. All
other things being equal, the lower an energy minimum governing the
stabilization of endotoxin in a given complex with an endotoxin
masker is, the more difficult it will be, i.e. the more stringent
the modulator must be, to liberate endotoxin from its solubilized
state. Yet as mentioned above, such liberation is an important step
in the eventual aggregation of endotoxin into a detectable, i.e.
unmasked, form. Thus, the more stable the complex between endotoxin
and endotoxin masker, the more rigorous must be the measures taken
to ultimately unmask the endotoxin.
In instances where the complex between endotoxin and endotoxin
masker is especially stable, addition of a single- or even
multiple-component modulator may sometimes not be enough to
destabilize the masking complex and liberate the endotoxin. It may
in such instances be helpful to promote endotoxin liberation from
its complex with endotoxin masker by adjusting solution conditions
so as to destabilize the endotoxin-endotoxin masker complex.
As mentioned above, an agent which influences hydrogen bonding
stability in solution may assist in this aim. Some, if not most of
the stabilization of endotoxin in complex with an endotoxin masker
normally arises from non-covalent interactions between the
endotoxin moiety and the endotoxin masker. These interactions may
for instance take the form of hydrophobic, ionic, hydrogen bonding
and/or Van der Waals interactions between regions of the endotoxin
molecule and regions on the molecule or molecules of the endotoxin
masker. As the strength of these endotoxin-endotoxin masker
interactions is influenced by the surrounding hydrogen bonding
network in solution, it conversely follows that influencing the
hydrogen bonding stability in solution will modulate the strength
of these interactions.
Addition of an agent which influences hydrogen bonding stability in
solution can therefore help to weaken the noncovalent bonding
interactions between endotoxin and endotoxin masker, essentially
raising the free energy of the complex and thus rendering it more
susceptible to disruption by the modulator so that the endotoxin is
liberated and rendered detectable.
Besides the destabilizing effect discussed above, an agent which
influences hydrogen bonding stability in solution may also have a
further effect promoting endotoxin unmasking. By altering hydrogen
bonding stability in solution, the agent may also foster
aggregation of the endotoxin moieties once liberated from their
complex with endotoxin masker. There will generally exist an
equilibrium between endotoxin in solubilized and aggregated forms.
The agent which influences hydrogen bonding stability in solution
can be helpful in shifting this equilibrium towards the aggregated
(and thus detectable). Suitable substances are those which would
tend to decrease the hydrogen bonding stability in solution
surrounding the chaperoned endotoxin moities, and/or compounds
which tend to increase the ionic strength of the solution, thus
driving the reconfiguring modulator-chaperoned endotoxin moieties
together into a lipophilic aggregate.
It should be noted that it may not always be necessary to add an
agent which influences hydrogen bonding stability in solution.
Whether or not addition of such an agent will be indicated will
depend, for instance, on the stability of endotoxin in complex with
the endotoxin masker and/or on the position of equilibrium between
solubilized, chaperoned and aggregated forms of endotoxin moieties
once liberated from the endotoxin masker. For instance, in
solutions containing higher concentrations of salt, it is
conceivable that the complex of the endotoxin and endotoxin masker
may already be instable enough to be broken up by the disrupting
modulator alone, and that the endotoxin moieties present in
solution following liberation from the endotoxin masker will be
instable enough so as to form aggregates without any further
assistance. In such situations, an agent which influences hydrogen
bonding stability in solution may not be required to achieve
unmasking.
On the other hand, there may exist situations, for instance in
solutions containing lower concentrations of salt, where the
endotoxin-endotoxin masker complex may be of such great stability
that a disrupting modulator alone cannot break it up to liberate
endotoxin, or where--even if liberated by disrupting modulator
alone--the equilibrium between solubilized and aggregated endotoxin
lies towards the solubilized form so that the aggregation needed
for detection does not occur. In such situations incorporation of
an agent which influences hydrogen bonding stability in solution
may help to influence the energetics of complexation and/or
aggregation so as to favor endotoxin in detectable form.
In general, it can be said that the degree of destabilization of
the complex between the endotoxin and endotoxin masker will depend
on the amount of salt in solution, with this complex being
destabilized to an extent directly proportional to the amount of
salt present in solution. As a general rule though, reference may
be made to the Hofmeister series, according to which the more
chaotropic a salt is, the lower the amount of such a salt will be
needed to destabilize a complex between endotoxin and endotoxin
masker to a given extent. Merely as an illustrative example, in
order to achieve approximately the same degree of destabilization
of a complex between endotoxin and endotoxin masker achievable
with, say, 100 mM CaCl.sub.2, one may need to use, say, 500 mM
NaCl. In this example, CaCl.sub.2 is more chaotropic than NaCl, so
less CaCl.sub.2 would be required to achieve the same degree of
destabilization.
In certain embodiments of the invention, the agent which influences
hydrogen bonding stability in solution may be a chaotropic agent, a
cation or a combination thereof. In certain embodiments, the
chaotropic agent may be chosen from the group consisting of urea,
guanidinium chloride, butanol, ethanol, lithium perchlorate,
lithium acetate, magnesium chloride, phenol, a propanol (e.g.
1-propanol or 2-propanol, i.e. isopropanol) and thiourea. In
certain embodiments, the cation is a divalent cation, for example
Ca.sup.2+, Mg.sup.2+, Sr.sup.2+ and/or Zn.sup.2+. An expecially
preferred divalent cation is Ca.sup.2+.
The agent which influences hydrogen bonding stability in solution,
e.g. CaCl.sub.2, may advantageously be used in a concentration
range of 1-400 mM, preferably in a concentration range of 10-200
mM, preferably at a concentration range of 50-100 mM in the
unmasking process.
Without being bound by theory, and merely to illustrate the
principles and possible mechanisms which the present inventors
believe underlie the observed advantageous effect of unmasking
endotoxin in solution, thereby rendering previously masked an
undetectable endotoxin detectable, the following describes several
mechanisms of interaction between endotoxin and further components
of a given composition containing at least one endotoxin masker. To
illustrate these mechanisms, reference is made to FIGS. 1-6.
Unmasking Endotoxin Masked by a Detergent Masker with a
Single-Component Modulator, in which the Single Component Functions
as Both a Disrupting Modulator and a Reconfiguring Modulator
FIG. 1 depicts the scenario in which endotoxin resides in solution
together with a detergent which is masking it in individualized
form in a detergent micelle. Panel (a) of FIG. 1 shows a single
endotoxin moiety which is inserted in the lipid layer of such a
detergent micelle via its lipid tail. The detergent molecules
constituting the lipid layer of the detergent micelle are
symbolized as open circles in panel (a). Because this single moiety
of endotoxin is stably inserted in individual form in the lipid
layer of the micelle rather than in multimeric, aggregated form, it
evades detection using available detection methods (e.g. the
EndoLISA.RTM. assay of Hyglos GmbH). If the solution shown in panel
(a) of FIG. 1 were a pharmaceutical formulation additionally
containing an API, it would appear to be endotoxin-free and
therefore safe for administration, even though endotoxin is present
in the solution. Administering such an ostensibly endotoxin-free
formulation to a patient would thus risk unwittingly eliciting the
types of dangerous immunological and toxic responses to endotoxin
mentioned above.
Above the equilibrium arrows between panels (a) and (b) of FIG. 1,
one sees the addition of a disrupting and reconfiguring modulator
capable of releasing the endotoxin from a complex between the
endotoxin and the endotoxin masker. In the scenario shown in in
FIG. 1, this "complex" is the endotoxin embedded, via its lipid
component, in the lipid layer of a detergent micelle. The
disrupting and reconfiguring modulator shown here (an amphiphilic
molecule used as a single-component modulator having capacity as
both a disrupting and reconfiguring modulator) exhibits the dual
properties of breaking up the detergent micelle so as to liberate
inserted molecules of endotoxin, as well as of stabilizing the
endotoxin once it is liberated from its complex with the endotoxin
masker. This latter characteristic is schematically depicted in the
upper portion of panel (b) of FIG. 1, showing a molecule of
endotoxin stabilized by the disrupting and reconfiguring modulator
such that the molecule of endotoxin can exist in chaperoned form
outside of the micelles once these are broken up by the modulator.
The lower portion of panel (b) makes clear that the disrupting
modulator exists in equilibrium, associated with both the liberated
endotoxin moiety and detergent previously making up the lipid layer
of the detergent micelle prior to the micelles disruption by the
disrupting (and reconfiguring) modulator.
As mentioned above, in one embodiment of the present invention, the
disrupting and/or reconfiguring modulator may be 1-dodecanol,
bearing a polar alcohol moiety, followed by a saturated hydrocarbon
tail of 12 carbon atoms. Both the steric and electrostatic
configuration of 1-dodecanol is thus similar to that of the lipid
moieties of the endotoxin, so that 1-dodecanol can efficiently
interact with, and therefore stabilize, the endotoxin after it has
been liberated from the detergent micelle.
Another reason why 1-dodecanol is especially suitable for use as a
disrupting and/or reconfiguring modulator is that 1-dodecanol,
although amphiphilic, does not form micelles. Thus, once the
detergent micelle depicted in panel (a) of FIG. 1 is broken up by
1-dodecanol, new micelles of modulator do not reform, which might
otherwise remask endotoxin by shifting equilibrium away from its
aggregated form. The characteristic of the modulator that it does
not form micelles itself thus contributes to the stabilization of
endotoxin in solution, aided by the modulator, as depicted in panel
(b) of FIG. 1. In the scenario depicted in FIG. 1, the hypothetical
prevailing solution conditions are such that equilibrium between
the chaperoned moieties of endotoxin shown in panel (b) and the
aggregated endotoxin shown in panel (c) already lies in the
direction of the aggregate of panel (c). With the aggregate form of
endotoxin favored, the endotoxin is already in, or predominantly in
an aggregated form which is amenable to detection by known means,
e.g. the EndoLISA.RTM. test kit of Hyglos GmbH.
Overall, then, FIG. 1 shows the transition from individual
endotoxin moieties (solubilized) which are stably inserted in and
therefore masked by detergent micelles to a scenario in which the
individual moieties of endotoxin have aggregated so as to become
detectable. Previously masked endotoxin in panel (a) has been
unmasked in panel (c), thereby allowing one to determine that a
solution previously thought to be free of endotoxin actually
contains this contaminant.
Unmasking Endotoxin Masked by a Detergent Masker with a
Dual-Component Modulator Comprising a Disrupting and Reconfiguring
Modulator and an Adsorbing Modulator (Protein)
The initial scenario depicted in FIG. 2 is much like that depicted
in FIG. 1: a single molecule of endotoxin is inserted in a
detergent micelle (symbolized by a ring of open circles
representing the individual detergent molecules) and, thus stably
individualized, is masked such that it evades detection. Between
panels (a) and (b), one sees the addition of a dual-component
modulator system comprising both a non-protein component
functioning simultaneously as a disrupting and reconfiguring
modulator and a protein component functioning as an adsorbing
modulator. The disrupting and reconfiguring modulator may be as
described as above for FIG. 1, e.g. 1-dodecanol, which helps to
disrupt the detergent micelle and stabilize/reconfigure the
liberated endotoxin, without forming micelles of its own. The
adsorbing modulator may for example be added as part of the
modulator in order to promote the disruption of detergent micelles
which are more stable than those depicted in FIG. 1, and for which
a disruption modulator alone may not suffice to achieve the desired
disruption.
As explained above, the adsorbing modulator may for instance be
bovine serum albumin (BSA) or human serum albumin (HSA), among
other things. Such proteins have the ability to act as "molecular
sponges" which adsorb on their surface molecules of the previously
micelle-forming detergent. Of course, in the event that such an
adsorbing modulator is employed, there will exist a certain
equilibrium between other detergent-like molecules in solution,
such as the disrupting and reconfiguring modulator. This would be
expected to engender an equilibrium as shown in panel (b), in which
the disrupting and reconfiguring modulator exists in forms bound to
liberated endotoxin (right portion of panel (b)), bound to
detergent previously constituting the detergent micelle, as well as
bound to the surface of the adsorbing modulator, along with
additional detergent from the (now disrupted) detergent
micelle.
Under the solution conditions prevailing in the scenario shown in
FIG. 2, endotoxin which has been liberated from the masking
detergent micelle combine into detectable aggregates, shown in
panel (c). In fact, the use of an adsorbing modulator as shown in
FIG. 2 can promote such aggregate formation. This is assumed to be
because the adsorbing modulator binds molecules of the disrupting
and reconfiguring modulator on its surface, thereby removing these
otherwise endotoxin-stabilizing species from solution such that
equilibrium is driven to the right toward the aggregate of panel
(c).
Overall, then, FIG. 2 shows the transition from individual
endotoxin moieties which are embedded in detergent micelles and,
due to their individualization in these micelles, remain masked, to
a scenario in which the individual moieties of endotoxin have been
forced to aggregate so as to become detectable. That is, previously
masked endotoxin in panel (a) has been unmasked in panel (c),
thereby allowing one to determine that a solution previously
thought (in panel (a)) to be free of endotoxin actually contains
this contaminant (panel (c)).
Unmasking Endotoxin Masked by a Detergent Masker with a
Multi-Component Modulator System in Combination with an Agent which
Influences Hydrogen Bonding Stability in Solution
In the scenarios depicted in FIGS. 1 and 2, the solution conditions
were such that use of a modulator system alone suffices to disrupt
masking detergent micelles. Looked at another way, neither of the
masking micelles of detergent shown in FIGS. 1 and 2 have been so
stable as to resist disruption using a disrupting modulator alone.
In addition, the conditions in FIGS. 1 and 2 have also been such
that the equilibria between the solubilized and aggregated forms of
endotoxin lay toward the aggregated form, so that detection of this
aggregated form was possible under the solution conditions shown
without any further measures needing to be taken.
The conditions underlying the scenario shown in FIG. 3 are now
different. Here, individual molecules of endotoxin are inserted in
the lipid layer of detergent micelles (again symbolized by a ring
of open circles representing the individual detergent molecules),
but whether due to solution conditions, the nature of the
interaction of the masking detergent with the endotoxin, or a
combination of these things, the endotoxin inserted in the
detergent micelle in panel (a) is more stable, and therefore less
resistant to disruption with disrupting modulator, than either of
the initial situations in FIGS. 1 and 2. Additional measures are
required to destabilize the detergent-endotoxin complex so that,
once destabilized, the modulator system can disrupt the micelle and
liberate the inserted endotoxin.
To this end, the scenario shown in FIG. 3 entails using an agent
which influences hydrogen bonding stability in solution, symbolized
by small squares added above the equilibrium arrows between panels
(a) and (b), and shown in their interaction with the
micelle-endotoxin complex in panel (b). As mentioned above, one
substance useful as an agent which influences hydrogen bonding
stability in solution is divalent calcium.
With the complex between the detergent masker and the masked
endotoxin thus destabilized, a modulator system comprising both an
adsorbing modulator and a displacing modulator is added (see above
equilibrium arrows between panels (b) and (c)) to displace the
endotoxin from the already destabilized micelle of masking
detergent. As mentioned above, the displacing modulator may be
sodium-dodecyl sulfate (SDS), itself a detergent. The possibility
that the modulator system contains a component which is itself a
detergent and which may form new micelles of its own, is
represented in panel (c) of FIG. 3 by a dotted circle, in which the
endotoxin is inserted. Under the conditions prevailing in FIG. 3,
however, any micelle formed by the displacing modulator is not as
stable as the micelle formed by the masking detergent shown in
panel (a). This is at least partly because the adsorbing modulator,
e.g. BSA shown in FIG. 3 also binds the displacing modulator on its
surface, establishing an equilibrium between protein-bound and
micelle-forming populations of the displacing modulator which
effectively destabilizes any micelle formed by the displacing
modulator.
The presence of a disrupting and reconfiguring modulator, for
instance a non-micelle-forming amphiphilic species such as
1-dodecanol, is shown over the equilibrium arrows between panels
(c) and (d) of FIG. 3. The remainder of the schematic shown in FIG.
3 is analogous to what has already been discussed in detail above
in the context of FIGS. 1 and 2. Briefly, the disrupting and
reconfiguring modulator shown between panels (c) and (d) of FIG. 3
liberates and solubilizes endotoxin transiently inserted in
micelles formed by the displacing modulator, at the same time
establishing an equilibrium between solubilized (non-detectable)
and aggregated (detectable) endotoxin species. This equlibrium may
be shifted to the right (toward aggregated form) by the agent which
influences hydrogen bonding stability in solution (e.g. a salt with
a cation, preferably a divalent cation and/or a chaotropic
agent).
Overall, FIG. 3 shows the liberation of a masked molecule of
endotoxin from a stable complex with a micelle of a detergent
masker. It uses an agent which influences hydrogen bonding
stability in solution to destabilize this complex, and a
multicomponent modulator which in total disrupts this complex and
chaperones the liberated endotoxin through a series of energetic
minima in the ultimate direction of an aggregated and therefore
detectable complex of endotoxin.
Unmasking Endotoxin Masked by a Protein Masker with a
Dual-Component Modulator Comprising a Displacing Modulator and a
Disrupting and Reconfiguring Modulator
FIG. 4 is a schematic depiction of a scenario in which an endotoxin
is masked by a protein in solution. This is shown in panel (a) of
FIG. 4. In the scenario depicted in FIG. 4, the protein, which may
for example be an API in a pharmaceutical formulation, exhibits a
binding cleft which is both sterically and electrostatically
suitable to stably bind endotoxin. In this way, the protein masker
binds molecules of endotoxin, rendering them undetectable. Addition
of a modulator component, symbolized by the displacing modulator
added above the equilibrium arrows between panels (a) and (b) of
FIG. 4, displaces the endotoxin from its binding site on the
protein masker. This displacing modulator might for instance be a
"second heteroatom-substituted aliphatic comprising 8 to 16 carbon
atoms" as discussed above. In the event that the displacing
modulator would be e.g. sodium dodecyl sulfate, this displacing
modulator might bind to the surface of the masking protein,
displacing the molecule of endotoxin from its stable binding
position within the protein's binding cleft. This is shown in the
left portion of panel (b) of FIG. 4. In addition, as symbolized by
the dotted circle in the right portion of panel (b), the displacing
modulator component may also form transient micelles of its own,
essentially chaperoning endotoxin liberated from the protein masker
in a form stably inserted into the micelle's lipid layer. The exact
position of the equilibrium shown in panel (b) of FIG. 4 depends on
the effectiveness with which the displacing modulator binds to the
surface of the masking protein (left portion of panel (b)), as well
as the stability of the micelle formed (right portion of panel
(b)).
Regardless of the exact position of this equilibrium, the important
thing is that the displacing modulator depicted above the
equilibrium arrows between panels (a) and (b) of FIG. 4 tends to
liberate the endotoxin from its energetically stable binding
position in or on the masking protein.
Once this endotoxin is freed from its masked state in or on the
masking protein, a further modulator component (disrupting and
reconfiguring modulator), depicted above the equilibrium arrows
between panels (b) and (c) of FIG. 4 shifts the energetic
relationships prevailing in solution such that the most stable
state for endotoxin is in freely solubilized form, chaperoned in
solution by the disrupting and reconfiguring modulator. This
disrupting and reconfiguring modulator may for example be a "first
heteroatom-substituted aliphatic comprising 8 to 16 carbon atoms"
as discussed above, which may for example be 1-dodecanol. As
discussed above, this disrupting and reconfiguring modulator will
typically have the property of disrupting existing micelles (for
example formed by the displacing modulator, and show in the right
portion of panel (b)), while not forming micelles of its own. With
any previous micelles of the displacing modulator thus disrupted,
and with the disrupting and reconfiguring modulator unable to form
corresponding micelles of its own, the most energetically stable
form of the endotoxin becomes the solubilized form shown in panel
(c) of FIG. 4, chaperoned by the disrupting and reconfiguring
modulator.
The remainder of FIG. 4 is as previously discussed for the final
equilibrium step in FIGS. 1 and 3. Briefly, there exists an
equilibrium between individual, solubilized endotoxin (panel (c))
and aggregated endotoxin (panel (d)). To the extent that any
appreciable population of aggregated endotoxin exists as part of
this equilibrium, the endotoxin becomes detectable where, stably
bound in or on the masking protein, it previously was not. Overall,
endotoxin which was previously masked in individualized form by a
protein has been unmasked and rendered detectable by adjusting the
solution conditions such that the most energetically favorable
state in which endotoxin can reside becomes its detectable
aggregated form. As in previous figures discussed above, then, the
"unmasking" is the result of manipulating solution conditions so as
to shift equilibrium from a state in which endotoxin is stabilized
in individualized form ("masked") toward a state in which endotoxin
is aggregated and detectable ("unmasked").
Unmasking Endotoxin Masked by a Protein Using a Multi-Component
Modulator Comprising an Adsorbing Modulator (Protein), a Displacing
Modulator and a Disrupting/Reconfiguring Modulator, in Combination
with an Agent which Influences Hydrogen Bonding Stability
The initial scenario shown in FIG. 5 corresponds to that shown in
FIG. 4: endotoxin is stably bound in or on a protein present in the
composition. This protein in the composition, which may for example
be an API, thus functions as an "endotoxin masker". As already
discussed in the context of the scenario depicted in FIG. 3, the
endotoxin is so stably complexed with the endotoxin masker in panel
(a) of FIG. 5 that simple addition of modulator cannot alone
liberate it. In FIG. 3, discussed above, the endotoxin masker was a
detergent, which formed a micelle in which a single molecule of
endotoxin was very stably inserted. Now in FIG. 5, the endotoxin
masker is a protein with a binding site amenable for stable
endotoxin binding. But the principle remains the same: Whether
inserted in the lipid layer of a detergent micelle (FIG. 3) or
residing stably in or on a protein, the endotoxin is stabilized to
an extent that simple addition of a modulator is unable to overcome
and the thus solubilized endotoxin remains undetectable.
As explained above for FIG. 3, this stable complex between
endotoxin and endotoxin masker can be destabilized by addition of
an agent which influences hydrogen bonding stability in solution,
for example a salt or a chaotropic agent, for example divalent
calcium. This agent which influences hydrogen bonding stability is
symbolized in FIG. 5 by small squares starting over the equilibrium
arrows between panels (a) and (b). This agent disrupts the hydrogen
bonding network which is assumed to exist between endotoxin and the
protein masker, thus raising the free energy of the complex to a
level where the modulator components, which are shown above the
equilibrium arrows between panels (b) and (c), can break up the
complex to such an extent that the endotoxin is dislodged from the
masking protein.
Using a modulator system comprising both an adsorbing modulator
(protein) and a displacing modulator as shown in FIG. 5 then is
assumed to lead to the equilibrium situation shown in panel (c). In
the left portion of panel (c) is the masking protein, now divested
of the endotoxin previously bound. Molecules of the agent which
influences hydrogen bonding stability in solution as well as of the
displacing modulator, for example SDS, are shown bound to the
surface of the masking protein, including in the binding site where
endotoxin was previously bound. This depiction is intended to
represent the fact that the displacing modulator effectively
displaced endotoxin from its stable position in or on the masking
protein. The middle portion of panel (c) of FIG. 5 shows a micelle
which might be formed by the displacing modulator (e.g. SDS), with
a molecule of endotoxin transiently inserted into the lipid layer
of the micelle. Molecules of the agent which influences hydrogen
bonding stability in solution are also shown bound to endotoxin and
micelle, and serve to further destabilize this micelle, ensuring
that the micelle in fact remains transient and does not present the
endotoxin with an energy binding minimum from which it cannot be
dislodged by a further disrupting modulator. Finally, the right
portion of panel (c) shows the adsorbing modulator (protein)
acting, as described briefly above, as a "molecular sponge" which
adsorbs both the agent which influences hydrogen bonding stability
in solution as well as the displacing modulator on its surface.
This effectively depletes these species in solution, destabilizing
the transient micelle shown in the middle portion of panel (c) to
the extent that the displacing modulator is depleted, while
stabilizing it to the extent that the agent which influences
hydrogen bonding stability in solution is depleted. Generally,
however, the amount of the agent which influences hydrogen bonding
stability in solution will be high enough to destabilize the
initial complex between masking protein and endotoxin that enough
of this agent will persist in solution despite depletion by the
adsorbing modulator, so that the transient micelle shown in panel
(c) will be destabilized as desired.
Use of a disrupting and reconfiguring modulator, for example as
shown over the equilibrium arrows between panels (c) and (d) of
FIG. 5 (e.g. 1-dodecanol), will then break up the transient micelle
shown in panel (c) so as to liberate the molecule of inserted
endotoxin. As already discussed above the thus solubilized
endototoxin (panel (d)) will then enter into an equilibrium
relationship with a reconfigured, aggregated form of endotoxin
(panel (e)) which can be detected as discussed above.
Unmasking Endotoxin Masked by Both Protein and Detergent Maskers
with a Multi-Component Modulator Comprising an Adsorbing Modulator
(Protein), a Displacing Modulator and a Disrupting/Reconfiguring
Modulator, in Combination with an Agent which Influences Hydrogen
Bonding Stability in Solution
Many protein APIs, for example, antibodies, antibody fragments,
hormones, enzymes, fusion proteins or protein conjugates are
formulated and marketed at such high concentrations that detergents
must be included in solution to avoid unwanted protein aggregation.
The initial scenario shown in FIG. 6 is thus representative of one
of the most relevant situations in the field of pharmaceutical
formulation because both detergent and protein (e.g. API protein)
maskers are present. The molecule of endotoxin is shown as inserted
in the lipid layer of a detergent micelle (again symbolized by a
ring of open circles representing the individual detergent
molecules) as well as bound in or on the masking protein. In
reality, these two species are likely to exist in equilibrium, with
the relative position of this equilibrium, toward either a micelle-
or a protein-bound species of endotoxin, being dictated by the
relative stability of the respective complexes. All other things
being equal, the complex of lower free energy, and therefore
greater stability will generally prevail.
The discussion of FIG. 6 is analogous to that of FIG. 5 above, with
the only difference being that panel (b) of FIG. 6 shows both the
protein- and micelle-bound species of endotoxin in mutual
equilibrium, each destabilized by the agent which influences
hydrogen bonding stability in solution. Using an adsorbing
modulator and a displacing modulator leads to the equilibrium
situation depicted in panel (c) of FIG. 6. The discussion above for
panel (c) of FIG. 5 applies here correspondingly. The use of a
further disrupting and reconfiguring modulator (shown over the
equilibrium arrows between panels (c) and (d)) which is capable of
disrupting the transient micelle of panel (c) without forming
micelles of its own, frees the endotoxin from its transiently bound
state in a micelle of displacing modulator (middle portion of panel
(c)), and engenders the equilibrium relationship between soluble
(non-detectable) and aggregated (detectable) forms of endotoxin as
discussed above. As explained above for previous figures, the
disrupting and reconfiguring modulator shown in panel (d) is shown
in equilibrium between states bound to the liberated endotoxin
(upper portion of panel (d)) and detergent previously constituting
the detergent micelle shown in panel (a) (lower portion of panel
(d)).
It should be noted that the above scenarios are intended to
illustrate the principles which the present inventors believe
underlie the advantageous unmasking effect of the present invention
in different situations. From the illustrative FIGS. 1-6, it will
be clear that the processes discussed are all equilibrium
processes, and that there is accordingly no prerequisite for the
order of addition of different components of the modulator system
or, if used, of the agent influencing hydrogen bonding stability
and solution. The equilibria shown will thus be automatically
established as soon as the components are present together in
solution. The "order" of addition of these components as implied in
the discussion above and shown in FIGS. 2-6 thus serves merely to
illustrate the mechanisms which the present inventors believe
underlie the advantageous technical effect of the present
invention. Accordingly, unmasking a previously masked endotoxin
might be accomplished by adding components at separate points in
time as suggested by FIGS. 2-6, however the desired unmasking
effect is also achievable when the components depicted in FIGS. 2-6
are added all at once.
In the most general sense, the scenarios depicted above in FIGS.
1-6 and the corresponding discussion should illustrate the
following general principles, which are intended as general
guidelines to the skilled person in implementing the present
invention. Many solutions which test negative for endotoxin by
conventional methods actually contain endotoxin in masked form.
Conventional methods detect endotoxin in its aggregated form, so
the fact that many existing solutions, such as pharmaceutical
formulations, test negative for endotoxin does not necessarily mean
that these solutions contain no endotoxin, but rather that they
contain no endotoxin in detectable form.
In their most general form, the methods of the invention allow
unmasking of endotoxin, e.g. by destabilizing complexes between
endotoxin and endotoxin maskers so as to liberate, and ultimately
aggregate individual molecules of endotoxin, thus rendering
previously undetectable endotoxin detectable. Liberation of
endotoxin from its masked complexes with endotoxin maskers may
ensue directly using a disrupting and reconfiguring modulator to
break up such complexes or, for especially stable complexes, these
may be destabilized and then broken up with such a modulator or
with a multi-component modulator system. However the bound
endotoxin is liberated, the net effect is that endotoxin
transitions from a stably bound form into a transient soluble form
which may then aggregate. In its broadest sense, then, the methods
of the present invention entail adjusting solution conditions as
described above so as to usher previously masked endotoxin through
a series of equilibria, wherein the final transition results in
aggregation of endotoxin in a form which is detectable.
Since the unmasking and/or detection of endotoxin according to the
methods described herein depend on a final reconfiguration of
liberated endotoxin in solubilized (undetectable) form into
aggregated form (detectable) a reconfiguring modulator will
generally be needed. This reconfiguring modulator (e.g.
1-dodecanol) will generally have the characteristic of not forming
micelles on its own, while stabilizing individual molecules of
endotoxin such that these can enter into an equilibrium with
aggregated forms of endotoxin. As is clear from the above, a
reconfiguring modulator will sometimes, but need not necessarily,
also function as a disrupting modulator which is able to break up
an initial complex between endotoxin and a micelle of masking
detergent and/or a complex of between endotoxin and a transient
micelle of displacing modulator.
The following examples, including the experiments conducted and the
results achieved, are provided for illustrative purposes only and
are not construed as limiting the present invention.
EXAMPLES
Introduction
Endotoxin masking is a common phenomenon in pharmaceutical
composition, especially biopharmaceutical drug products. Masking of
endotoxin is driven by several factors, leading in the end to the
non-detectability or at least a decreased detectability of the
endotoxin in the drug product.
In one scenario, masking is not caused by the active pharmaceutical
ingredient (API), e.g. protein, itself but by the formulation
ingredients. Such ingredients are detergents, which are added to
prevent aggregation of the protein, and buffer substances like
citrate, phosphate, Tris, acetate, histidine, glycine which are
added for pH-adjustment of the product.
Unsurprisingly, the kinetics of masking is influenced by
temperature, with masking proceeding faster at higher temperatures
than at lower temperatures. Unless otherwise specified, all
experiments described below were performed at room temperature.
This is the temperature at which production process steps of the
active pharmaceutical ingredient (API) are often performed, and is
therefore the most relevant temperature for assessing the
applicability of the inventive methods described herein to
industrial processes.
Example 1: Unmasking of Endotoxin from a Masking System of
Polysorbate 20/Citrate Using a Disrupting and Reconfiguring
Modulator (1-Dodecanol) Alone, and Together with a Further
Adsorbing Modulator (BSA)
A masking system of polysorbate 20/citrate was chosen for the first
experiment because citrate and polysorbate 20 are often included in
biopharmaceutical formulations. These experiments are intended to
determine whether masked endotoxin can be released from a complex
with detergent masker by addition of a disrupting and reconfiguring
modulator as described herein.
Materials and Methods
Endotoxin masking was performed as follows. 1 ml aqueous aliquots
of 10 mM Citrate pH 7.5 containing 0.05% (w/v) of polysorbate 20
were prepared in endotoxin-free glass test tubes. Subsequently, 10
.mu.l of a 10,000 EU/ml LPS stock solution (LPS 055 B5, Sigma
L2637-5MG) were added, the resulting solution was vortexed for 1
min and was stored at room temperature for at least 24 hours. As a
positive LPS control containing non-masked LPS, 10 .mu.l of a
10,000 EU/ml LPS stock solution was added to 1 ml of endotoxin-free
water, mixed and identically incubated as the masking preparations,
but without polysorbate 20. The LPS-water positive control is
described in more detail below.
Endotoxin unmasking was performed as follows. 100 .mu.l of stock
solutions of each of 1-dodecanol (disrupting and reconfiguring
modulator) dissolved in 100% ethanol and 100 mg/ml BSA (adsorbing
modulator) dissolved in endotoxin-free water were added.
1-dodecanol and BSA are used here as the two components of a
dual-component modulator system. A separate unmasking experiment
was performed identically as above, except that a single-component
modulator was used. The single modulator in this experiment was
1-dodecanol alone, i.e. without BSA. Concentrations of the
1-dodecanol stock solutions were 400, 200, 100, 50, 25, 12.5 and
6.25 mM. For unmasking, the unmasking stock solutions of BSA and
1-dodecanol were sequentially added with 2 minutes mixing by
vortexing after each addition. After mixing, the samples were
incubated for 30 minutes at room temperature without mixing.
Endotoxin content was analyzed using EndoLISA.RTM. (Hyglos GmbH)
according to the kit instructions. Sample dilutions were 1:10 and
1:100 in endotoxin-free water.
Endotoxin recovery was calculated as a percentage of recovery of a
separate LPS-water control containing only water and LPS without
any masking component. In the absence of any endotoxin masker, no
LPS in this LPS.water control should be masked, that is all LPS
present in this LPS-water control should be detectable. In this
way, the LPS-water control serves as a standard to determine both
qualitatively as well as quantitatively whether the EndoLISA.RTM.
detection kit employed is functioning properly to detect LPS
(qualitative control), and whether all LPS known to be present in
the control is in fact detected (quantitative control).
Results
The recovery data in FIG. 7 and Table 1 (below) show that by the
addition of BSA and/or 1-dodecanol in concentrations from 20 to 2.5
mM, masked endotoxin can be recovered to an extent greater than
100%. In the absence of BSA, 100% recovery cannot be achieved but,
rather, greater than 50% in the range of 10 to 2.5 mM of
1-dodecanol with maximum recovery at 5 mM 1-dodecanol of
approximately 90%.
In this and following examples, recoveries of greater than 100% of
LPS should be interpreted in light of the following: The activity
of LPS has been found to depend on both LPS form (e.g. extent and
orientation of aggregation) as well as LPS structure (this
structure varying slightly in LPS deriving from different bacterial
species). The inventive unmasking methods described herein have the
potential to alter both the form and the orientation of LPS
aggregation (indeed, it is due to such alteration as promoted by
the modulator, especially the reconfiguring modulator, that
unmasking of LPS is possible at all). The change in form and
orientation of LPS aggregation between the LPS-water control (not
unmasked) and the unmasked samples may in some cases cause the
activity detected following unmasking to exceed that measured in
the positive LPS-water control. This does not mean that performing
the inventive unmasking methods as described herein generates new
LPS not previously present, but rather than in some cases,
performing the inventive unmasking methods as described herein
alter the form of existing LPS such that the apparent measured
activity for a given amount of LPS increases.
TABLE-US-00001 TABLE 1 1-Dodecanol (mM) BSA (mg/ml) % LPS recovery
40 -- 28 20 -- 46 10 -- 60 5 -- 89 2.5 -- 65 1.25 -- 31 0.625 -- 7
40 10 70 20 10 157 10 10 186 5 10 170 2.5 10 134 1.25 10 71 0.625
10 0
The results clearly demonstrate that masked endotoxin can be
unmasked by the addition of the modulator 1-dodecanol (disrupting
and reconfiguring modulator) alone. The results further show that
this unmasking effect can be improved by the addition of a further
adsorbing modulator (BSA). In this latter case in which 1-dodecanol
and BSA are added as a dual-component modulator, the BSA helps to
adsorb detergent, thus destabilizing the detergent micelle masking
the endotoxin, the modulator 1-dodecanol, is capable of disrupting
detergent micelles (in its capacity as disrupting modulator) and
reconfiguring liberated endotoxin into an aggregate structure (in
its capacity as reconfiguring modulator). In the case of
polysorbate 20 in the absence of BSA an almost quantitative
recovery is possible (89% at 5 mM 1-dodecanol). This may be due to
the similarity in the length of the alkyl chains of 1-dodecanol and
the LPS-masking detergent polysorbate 20. The unmasking is improved
by the addition of BSA, which is assumed to shift the equilibrium
of LPS from solubilized to aggregated form (see e.g. FIG. 2).
Example 2: Unmasking of Endotoxin from a Masking System of
Polysorbate 20/Citrate Using Alcohols of Different Alkyl Chain
Length as Disrupting and Reconfiguring Modulators
This experiment investigates the use of various alkyl alcohols as
disrupting and reconfiguring modulators. One aim of the experiments
described in this example was to investigate the relationship
between alkyl chain length in the alcohol and unmasking efficiency.
To this end, unmasking was performed by the addition of alcohols
with carbon atom chain lengths from C8-C18 in different
concentrations.
Materials and Methods
Endotoxin masking was performed as described in Example 1.
Unmasking was performed by the addition of stock solutions of
unbranched 1-alcohols of different alkyl chain lengths (C.sub.8,
Co, C.sub.12, C.sub.14, C.sub.16, C.sub.18) as modulators
(disrupting and reconfiguring modulators) as described in Example 1
for 1-dodecanol (having a 12-carbon alkyl chain). Each of the stock
solutions was dissolved in 100% ethanol. In contrast to certain of
the experiments described above in Example 1, no other modulator
components, e.g. BSA, were included in the present unmasking
experiments. Analysis of endotoxin concentrations was performed
using the EndoLISA.RTM. kit (Hyglos GmbH), and the subsequent
calculation of endotoxin recovery was expressed as a percent of the
LPS in the LPS-water control sample. The LPS-water positve control
is explained in detail in Example 1, above.
Results
Table 2 (below) show the percentage of unmasked endotoxin as
dependent on alcohol concentration and the length of the alkyl
chain in the alcohol.
TABLE-US-00002 TABLE 2 % LPS Recovery 1- 1- Conc. 1- 1- 1- 1- Hexa-
Octa- (mM) Octanol Decanol Dodecanol Tetradecanol decanol decanol
40 0 0 28 10 nd nd 20 0 0 46 36 1 1 10 0 0 60 44 3 1 5 0 1 89 28 3
0 2.5 0 5 65 16 0 3 1.25 0 1 31 25 0 1 0.625 1 4 7 10 2 1 nd = no
data
Endotoxin recoveries of, i.e. unmasking endotoxin by, greater than
40% were achieved using 1-dodecanol and 1-tetradecanol. Recoveries
using alcohols with alkyl chains lengths below or above C12 and C14
are below 10%.
The above results imply that the alkyl chain length of the alcohol
used as a disrupting and reconfiguring modulator should ideally
match the alkyl chain length of the acyl chains in the endotoxin as
closely as possible. In the present case, the lengths of the acyl
chains in the Lipid A component of LPS are C12 and C14, and it was
the 1-alcohols having alkyl chain lengths in that range which, when
used as disrupting and reconfiguring modulators, most effectively
unmasked the endotoxin.
Example 3: Unmasking of Endotoxin from Masking Systems of Various
Non-Ionic Surfactants Using 1-Dodecanol as a Disrupting and
Reconfiguring Modulator Alone, and Together with the Adsorbing
Modulator BSA
To investigate the hypothesis that unmasking endotoxin from
polysorbate 20 by 1-dodecanol alone is promoted by equivalent or
similar alkyl chain length of the masking surfactant and
1-dodecanol, various experiments were designed using masking
detergents of different chain lengths and different structure, and
these were then unmasked using a disrupting and reconfiguring
modulator of fixed alkyl chain length (1-dodecanol, with a C.sub.12
alkyl chain). To this end, masked samples were prepared in
polysorbate 80 and TRITON.TM. (non-ionic surfactant) X-100 and
these were subsequently unmasked with 1-dodecanol or
BSA/1-dodecanol using different concentrations of 1-dodecanol.
Materials and Methods
Endotoxin masking was performed as follows: 1 ml aliquots of 10 mM
citrate pH 7.5 containing 0.05% of polysorbate 20, polysorbate 80
or TRITON.TM. (non-ionic surfactant) X-100 were prepared in
endotoxin-free glass test tubes. Subsequently, 10 .mu.l of a 10,000
EU/ml LPS stock solution (LPS 055 B5, Sigma L2637-5MG) were added,
vortexed for 1 min and stored at room temperature for at least 24
hours. As a positive LPS control, 10 .mu.l of a 10,000 EU/ml LPS
stock solution was added to 1 ml of endotoxin-free water, mixed and
identically incubated as the masking preparations. The positive
LPS-water control is discussed in detail above in Example 1.
Unmasking was performed by the addition of stock solutions of
1-dodecanol (as a disrupting and reconfiguring modulator) in
different concentrations as described in Example 1. Stock solutions
of the respective alcohols were dissolved in 100% of ethanol.
Unmasking was performed in both the absence and presence of 10
mg/ml BSA as described in Example 1. Analysis of endotoxin
concentrations was performed with the EndoLISA.RTM. kit (Hyglos
GmbH), with subsequent calculation of recovery of endotoxin
expressed as a percent of the endotoxin in the LPS/water control
sample.
Results
Table 3 (below) shows the recoveries of LPS after unmasking from
the respective polysorbate 20/citrate, polysorbate 80/citrate and
TRITON.TM. (non-ionic surfactant) X-100/citrate masking systems as
dependent on the 1-dodecanol (disrupting and reconfiguring
modulator) concentration in the absence or presence of BSA
(adsorbing modulator).
TABLE-US-00003 TABLE 3 Dodecanol BSA % LPS recovery (mM) (mg/ml)
Polysorbate 20 Polysorbate 80 Triton X-100 40 -- 28.0 4.9 nd 20 --
46.2 7.5 3.4 10 -- 60.5 11.5 nd 5 -- 89.1 25.2 0.0 2.5 -- 64.9 28.5
nd 1.25 -- 31.2 12.1 0.0 0.625 -- 7.2 0.0 nd 0.313 -- nd nd 0.0 40
10 69.7 19.4 nd 20 10 156.8 36.4 2.0 10 10 186.1 69.9 nd 5 10 170.5
86.9 23.0 2.5 10 133.5 94.2 nd 1.25 10 71.3 2.9 0.0 0.625 10 0.0
12.9 nd 0.313 10 nd nd 0.0 nd = no data
Unmasking with 1-dodecanol from the polysorbate 80/citrate masking
system results in recovery of approximately 30% at an optimal
concentration of 1-dodecanol of 2.5 mM. In the presence of BSA up
to 90% can be recovered. Both unmasking approaches from the
TRITON.TM. (non-ionic surfactant) X-100 masking system (i.e. with
and without BSA) result in LPS recoveries below 20%, regardless of
the concentration of 1-dodecanol.
Thus, unmasking using 1-dodecanol alone (as a disrupting and
reconfiguring modulator) is sufficient to unmask LPS from masking
systems such as in the polysorbate 20 masking system. The addition
of BSA (as an adsorbing modulator) to adsorb the masking detergent
improves unmasking recoveries in the polysorbate 20 and polysorbate
80 masking systems. Unmasking from the TRITON.TM. (non-ionic
surfactant) X-100 system is not highly efficient even when BSA is
added together with 1-dodecanol. Adding a further modulator
component such as e.g. SDS (as a displacing modulator) can help
improve recovery of LPS from TRITON.TM. (non-ionic
surfactant)-X-100 masking formulations.
Example 4: Increasing Unmasking Efficiency by Addition of a
Modulator and a Chaotropic Agent which Influences Hydrogen-Bonding
Stability
The weak recovery of LPS from the TRITON.TM. (non-ionic surfactant)
X-100 masking system using the dual-modulator system of BSA
(adsorbing modulator) and 1-dodecanol (disrupting and reconfiguring
modulator) may be due to the high stability of the complex formed
by TRITON.TM. (non-ionic surfactant) X-100 and LPS. This high
stability may prevent the desired destruction of the
endotoxin-masking micelles of TRITON.TM. (non-ionic surfactant)
X-100 by the disrupting action of 1-dodecanol and adsorption of the
detergent by BSA.
For this reason, the present experiments investigate the
possibility of destabilizing the masking complex by addition of a
chaotropic salt together with a multi-component modulator. The hope
was that by destabilizing an otherwise stable detergent micelle,
destruction of this micelle using a multi-component modulator
system of 1-dodecanol (as disrupting and reconfiguring modulator),
BSA (as adsorbing modulator) and SDS (as displacing modulator)
would then become possible.
Materials and Methods
Endotoxin masking was performed as follows: 1 ml aliquots of 10 mM
citrate pH 7.5 containing 0.05% of TRITON.TM. (non-ionic
surfactant) X-100 were prepared in endotoxin-free glass test tubes.
Subsequently, 10 .mu.l of a 10,000 EU/ml LPS stock solution (LPS
055 B5, Sigma L2637-5MG) were added, vortexed for 1 min and stored
at room temperature for at least 24 hours. As a positive LPS
control, 10 .mu.l of a 10,000 EU/ml LPS stock solution was added to
1 ml of endotoxin free water, mixed and incubated in an identical
manner as the masking preparations. The positive LPS-water control
is discussed in detail above in Example 1.
Unmasking endotoxin was performed as follows: 100 .mu.l of the
following stock solutions were added as single component or as
combinations to the 1 ml masked samples: 1 M CaCl.sub.2 (dissolved
in water), 100 mg/ml BSA (dissolved in water), 1% SDS (dissolved in
water) and 50 mM 1-dodecanol (dissolved in 100% ethanol). In the
case of addition of combinations, the agents were added
sequentially, with a 2-minute vortexing step between each addition.
The samples were then incubated at room temperature for 30 minutes
without shaking.
Endotoxin content was analyzed using the EndoLISA.RTM. kit (Hyglos
GmbH) according to the kit instructions. Sample dilutions were 1:10
and 1:100 in endotoxin-free water. Endotoxin recovery was
calculated and expressed as a percentage of recovery of the
LPS-water control. The positive LPS-water control is discussed in
detail above in Example 1.
Results
FIG. 8 shows the percentage of LPS recovery as dependent on the
addition of combinations of CaCl.sub.2 (C), BSA (B; adsorbing
modulator), SDS (S; displacing modulator) and 1-dodecanol (D;
disrupting and reconfiguring modulator). 1-Dodecanol as the sole
(disrupting and reconfiguring) modulator does not efficiently
unmask LPS from a TRITON.TM. (non-ionic surfactant) X-100 masking
complex. Addition of BSA (adsorbing modulator) and 1-dodecanol
(disrupting and reconfiguring modulator) as a dual-component
modulator system results in approximately 20% recovery. Further
addition of either a chaotropic salt such as CaCl.sub.2 or a
further modulator such as SDS (displacing modulator) does not
result in LPS recoveries greater than 20%. However, the addition of
CaCl.sub.2, BSA (adsorbing modulator), SDS (displacing modulator)
and 1-dodecanol (disrupting and reconfiguring modulator) results in
LPS recoveries of greater 100%.
Thus, additionally to BSA (adsorbing modulator) and 1-dodecanol
(disrupting and reconfiguring modulator), a chaotropic salt and a
further displacing modulator such as the detergent SDS help to
break up the TRITON.TM. (non-ionic surfactant) X-100 masking
complex. In this way, the combination of these 4 additives seems to
break apart the masking complex and allows the formation of
detectable LPS.
Example 5: Comparison of Different Unmasking Approaches from
Various Masking Systems
As efficient unmasking from the TRITON.TM. (non-ionic surfactant)
X-100 masking system was observed using a combination of
CaCl.sub.2, BSA, SDS and 1-dodecanol, the question of unmasking
efficiency of this approach starting from polysorbate masking
systems remains. To answer this question, endotoxin was masked in
polysorbate 20, 80 and TRITON.TM. (non-ionic surfactant)
X-100/citrate masking systems and subsequently unmasked using
1-dodecanol alone; BSA and 1-dodecanol in combination; or
CaCl.sub.2, BSA, SDS and 1-dodecanol in combination. In these
experiments, 1-dodecanol is used as a disrupting and reconfiguring
modulator, BSA is used as an adsorbing modulator, SDS is used as a
displacing modulator and CaCl.sub.2 is used as an agent which
influences hydrogen-bonding stability in solution.
Materials and Methods
Endotoxin masking was performed as follows: 1 ml aliquots of 10 mM
citrate pH 7.5 containing either 0.05% polysorbate 20, or 0.05%
polysorbate 80 or 0.05% TRITON.TM. (non-ionic surfactant) X-100
were prepared in endotoxin-free glass test tubes. Subsequently, 10
.mu.l of a 10,000 EU/ml LPS stock solution (LPS 055 B5, Sigma
Aldrich L2637-5MG) were added, vortexed for 1 min and stored at
room temperature for at least 24 hours. As a positive LPS control,
10 .mu.l of a 10,000 EU/ml LPS stock solution were added to 1 ml of
endotoxin-free water, mixed and identically incubated as the
masking preparations. The function of the positive LPS-water
control is as described above in Example 1.
Unmasking of endotoxin was performed as follows: Either 100 .mu.l
of a 50 mM 1-dodecanol stock solution; or 100 .mu.l of 100 mg/ml
BSA and 100 .mu.l of a 50 mM 1-dodecanol stock solution; or 100
.mu.l of a 1 M CaCl.sub.2 solution, 100 ml of a 100 mg/ml BSA
solution, 100 .mu.l of a 1% SDS solution and 100 .mu.l of a 50 mM
1-dodecanol solution were added to the solution containing masked
LPS. In the case of addition of combinations, the agents were added
sequentially with a 2-minute vortexing step between each addition.
The samples were then incubated at room temperature for 30 minutes
without shaking.
Endotoxin content was analyzed using the EndoLISA.RTM. kit (Hyglos
GmbH) according to the kit instructions. Sample dilutions were 1:10
and 1:100 in endotoxin-free water. Endotoxin recovery was
calculated as a percentage of recovery of the LPS-water
control.
Results
Table 4 (below) and FIG. 9 show the percentages of LPS recovery
using either 1-dodecanol alone; BSA and 1-dodecanol in combination;
or CaCl.sub.2, BSA, SDS and 1-dodecanol in combination (CBSD) for
unmasking from various detergent masking systems.
TABLE-US-00004 TABLE 4 % LPS recovery Masking detergent 1-dodecanol
BSA/1-dodecanol CBSD Polysorbate 20 78 170 141 Polysorbate 80 28 94
161 TRITON .TM. (non- 0 23 168 ionic surfactant) X-100
Efficient (.about.80%) unmasking from the polysorbate 20 masking
system is achieved by 1-dodecanol, BSA/1-dodecanol and
CaCl.sub.2/BSA/SDS/1-dodecanol. In the case of a polysorbate 80
masking system, good unmasking efficiency is achieved in the
presence of BSA/1-dodecanol and CaCl.sub.2/BSA/SDS/1-dodecanol. In
the case of a TRITON.TM. (non-ionic surfactant) X-100 masking
system, the addition of CaCl.sub.2/BSA/SDS/1-dodecanol results in
good LPS recovery.
Thus, dependent on the stability of the masking complex, efficient
endotoxin recoveries can be achieved using different unmasking
approaches. However, the unmasking approach involving the
combination of CaCl.sub.2, BSA, SDS and 1-dodecanol may be the most
universal method, due to its ability to achieve efficient
unmasking, regardless of the masking system used. As is clear from
the experiments described herein above, an optimal composition for
unmasking LPS in any given formulation can be easily achieved by
routine experimentation.
Example 6: Unmasking of Endotoxin from Different Endotoxin
Sources
Endotoxin unmasking experiments in Examples 1-5 were performed with
a commercially available, highly purified LPS preparation of E.
coli O55:B5. As only the conserved Lipid A part of LPS is
responsible for toxicity and for detectability in Factor C-based
detection methods, it can be assumed that the unmasking approaches
described above will work equally well using LPS preparations from
bacteria other than E. coli O55:B5. However, the literature also
describes differences in acyl chain length for the lipid A part of
LPS, as well as modifications of side chains. Even more, the length
of the O-sugar side chains of LPS could potentially impact the
unmasking approach. Furthermore, it cannot be excluded that
purified LPS and naturally occurring endotoxin (NOE) may differ in
their unmasking behavior. To address these issues, and exclude the
possibility, that the unmasking approaches are specific for the
used LPS of E. coli O55:B5, LPS from different bacteria, different
length in core- and O-sugar chains and different purity were masked
in various detergent masking systems and subsequently unmasked
using either 1-dodecanol alone, BSA/1-dodecanol or
CaCl.sub.2/BSA/SDS/1-dodecanol.
Materials and Methods
Masking of endotoxin was performed as follows: LPS samples of
different types and from different sources were (approximately 50
EU/mL) added to 1 ml masking samples containing either 0.05%
polysorbate 20, 0.05% polysorbate 80 or 0.05% TRITON.TM. (non-ionic
surfactant) X-100 and 10 mM citrate pH 7.5. LPS source, type and
the supplier are shown in Table 5 (below). NOEs were produced from
bacterial culture supernatant after growth to stationary phase in
LB media by sterile filtration. As a preservative, 0.05% sodium
azide was added. Lyophilized LPS was dissolved in endotoxin-free
water. LPS solutions for which the supplier in Tables 5-7 is
indicated as "LMU" were kind gifts of Dr. A. Wieser of the
Ludwig-Maximilian University of Munich. Endotoxin content of the
LPS stock solutions was determined using the EndoZyme.RTM. kit
(Hyglos GmbH) and stock solutions of approx. 5000 EU/ml LPS in
endotoxin-free water were produced. From these solutions 10 .mu.l
were added to 1 ml masking samples. Afterwards, the samples were
allowed to mask the respective LPS for 7 days at room
temperature.
Unmasking of endotoxin was performed by addition of 100 .mu.l of
either a 100 mM 1-dodecanol stock solution, or addition of 100
.mu.l of a 100 mg/ml BSA and 100 .mu.l of 100 mM 1-dodecanol stock
solution or by addition of 100 .mu.l of each of 1 M CaCl.sub.2),
100 mg/ml BSA, 1% SDS and 100 mM 1-Dodecanol solutions. Unmasking
and determination of endotoxin content were performed as described
in Examples 1-5.
Results
Tables 5-7 (below) show the percent of LPS recovery after masking
and after unmasking of LPS from different sources and typs out of
different detergent masking systems. Specifically, Table 5 shows
the results obtained for a masking system of TWEEN 20.TM.
(polysorbate 20)/Citrate; Table 6 shows the results obtained for a
masking system of TWEEN 80.TM. (polysorbate 80)/Citrate; and Table
7 shows the results obtained for a masking system of TRITON.TM.
(non-ionic surfactant) X-100/Citrate.
TABLE-US-00005 TABLE 5 Tween 20/Citrate masking system Masking
CaCl.sub.2/BSA/SDS/ control Dodecanol BSA/Dodecanol Dodecanol
supplier (% recovery) (% recovery) (% recovery) (% recovery)
Klebsiella LMU 0.0 66 128 212 pneumonia Morganella LMU 0.0 81 110
120 morganii Yersinia LMU 0.0 63 174 243 enterocolitica Serratia
LMU 0.0 128 168 182 marcescens Neisseria LMU 0.0 9 23 38 meningitis
Acinetobacter LMU 0.0 0 124 655 baumanni * Enterobacter Hyglos 0.0
55 156 187 cloacae (NOE) * Salmonella Sigma 0.0 42 63 76 enterica
E. coli K 12 Invivogen 3.0 78 80 137 Pseudomonas Sigma 0.0 14 5 179
aeruginosa * * Strains which are common water contaminants, and
therefore more likely to be present in processes for the production
of pharmaceutical compositions
TABLE-US-00006 TABLE 6 Tween 80/Citrate masking system Masking
CaCl.sub.2/BSA/SDS/ control Dodecanol BSA/Dodecanol Dodecanol
supplier (% recovery) (% recovery) (% recovery) (% recovery)
Klebsiella LMU 0.0 12 173 353 pneumonia Morganella LMU 15.0 15 39
99 morganii Yersinia LMU 7.0 22 168 309 enterocolitica Serratia LMU
0.0 105 199 326 marcescens Neisseria LMU 0.0 0 11 42 meningitis
Acinetobacter LMU 0.0 7 337 511 baumanni * Enterobacter Hyglos 24.2
27 74 183 cloacae (NOE) * Pseudomonas Sigma 1.0 1 1 90 aeruginosa *
Salmonella Sigma 0.0 18 10 69 enterica E. coli K 12 Invivogen 1.9
85 106 176 * Strains which are common water contaminants, and
therefore more likely to be present in processes for the production
of pharmaceutical compositions
TABLE-US-00007 TABLE 7 Triton X-100/Citrate masking system Masking
CaCl.sub.2/BSA/SDS/ control Dodecanol BSA/Dodecanol Dodecanol
supplier (% recovery) (% recovery) (% recovery) (% recovery)
Klebsiella LMU 9.8 22 12 162 pneumonia Morganella LMU 5.5 35 23 48
morganii Yersinia LMU 0.0 13 19 236 enterocolitica Serratia LMU 3.5
28 20 80 marcescens Neisseria LMU 0.0 55 14 161 meningitis
Acinetobacter LMU 7.8 0 57 918 baumanni * Enterobacter Hyglos 0.0 2
26 85 cloacae (NOE) * Pseudomonas Sigma 0.0 1 11 25 aeruginosa *
Salmonella enterica Sigma 0.0 21 12 234 * Strains which are common
water contaminants, and therefore more likely to be present in
processes for the production of pharmaceutical compositions
The above data clearly show that the ability to successfully unmask
endotoxin from various masking systems is independent of the source
and type of LPS used. These results are important because they show
that the unmasking methods of the present invention represent a
general teaching applicable to various types of endotoxin from
various sources, under a variety of masking conditions.
Example 7: Unmasking of Endotoxin from Protein Masking Systems
The previous experiments have investigated the unmasking of LPS
from detergent masking systems. However, as described herein above,
detergents are not the only substances which can mask endotoxin
from detection. Proteins (e.g. protein APIs) are also capable of
masking endotoxin from detection when they contain binding sites on
or within their structure in which endotoxin can bind, thus evading
detection. The present experiments therefore relate to the masking
of endotoxin (LPS) by a protein rather than a detergent. Lysozyme
was used as the masking protein in these experiments because its
ability to bind endotoxin is known (see e.g. Ohno & Morrison
(1999). J. Biol. Chemistry 264(8), 4434-4441).
Materials and Methods
Endotoxin masking was performed as follows: 50 EU/ml of LPS (E.
coli O55:B5) was incubated for seven days in 10 mM citrate buffer,
pH 7.5 containing 1 mg/ml hen egg white lysozyme (Sigma Aldrich) at
room temperature.
Endotoxin unmasking was performed as follows: Unmasking was
performed by addition of unmasking reagents (modulators as
described in previous examples and agents influencing hydrogen
bonding stability) in various combinations. Specifically, 100 .mu.l
of the following unmasking agents were added to 1 ml aliquots of
the masked samples: 1-dodecanol, CaCl.sub.2, BSA, SDS. All stock
solutions were dissolved in water except 1-dodecanol, which was
dissolved in 100% ethanol. The added concentrations of the stock
solutions were 100 mM 1 M CaCl.sub.2, 100 mg/ml BSA and 1% SDS,
respectively. Unmasking was performed by sequential addition of the
various components with a two-minute vortexing step after each
addition. The samples were then incubated for 30 minutes at room
temperature and subsequently diluted 1:10 and 1:100 in
endotoxin-free water for analysis using the EndoLISA.RTM. kit
(Hyglos GmbH).
Results
Table 8 (below) shows the efficiency unmasking from a protein
masker (lysozyme) as dependent on the added components.
TABLE-US-00008 TABLE 8 CaCl2 BSA SDS 1-dodecanol % recovery LPS - -
- - 0 + - - - 0 + + - - 4 + + + - 33 + + + + 115 + + - + 15 + - + +
0 + - + - 4 + - - + 2 - + - - 9 - + + - 0 - + + + 1 - + - + 6 - - +
- 0 - - + + 0 - - - + 1
In the case of masking by lysozyme, use of 1-dodecanol
(reconfiguring modulator) alone or together with a supporting
detergent (displacing modulator) as a further component of the
modulator system does not efficiently unmask. Here, the
lysozyme-LPS masking complex seems to be more stable due to
electrostatic interactions between the negatively charged LPS and
the positively charged lysozyme. Improvement of unmasking may be
achieved by the addition of salt, which disrupts the electrostatic
interaction, thus rendering the lysozyme-LPS complex more labile
and increasing its susceptibility to disruption with modulator. To
this end, good results may be achieved by using a multi-component
modulator system of BSA (adsorbing modulator), SDS (displacing
modulator) and 1-dodecanol (reconfiguring modulator), together with
CaCl.sub.2 to lower the stability of the initial lysozyme-LPS
complex. The combination of these components is able to break up
the masking complex and lead to detectable LPS structures. This
model may be taken as a general model of the measures which may be
used to unmask endotoxin when it is masked, in whole or in part, by
a protein, e.g. a protein API in a pharmaceutical composition.
Example 8: Substances Other than 1-Alkyl Alcohols as Modulators for
Unmasking
As described herein above, 1-alkyl alcohols (used as reconfiguring
modulators) have been found to promote the formation of detectable
LPS structures. It was therefore desired to investigate whether
other types of substances than 1-alkyl alcohols might also have the
ability to promote similarly detectable forms of LPS. This example
shows the results of a screening for substances other than
1-alkyl-alcohols which might be able to support formation of
detectable LPS structures.
Materials and Methods
LPS (E. coli O55:B5, Sigma) 100 EU/ml was masked in polysorbate
20/citrate for 24 hours at room temperature. Unmasking was
initiated by sequential addition of 1 part stock solutions of
CaCl.sub.2 (at 1 M), BSA (at 100 mg/mL), SDS (at 1%) and substance
X into 10 parts of a solution of masked LPS, wherein "substance X"
represented the substance other than a 1-alkyl alcohol, the ability
of which as a reconfiguring modulator was to be tested. Substance X
was titrated in different concentrations. After unmasking, samples
were diluted 1:10 and 1:100 in endotoxin-free water and analyzed
for detectable endotoxin using the EndoLISA.RTM. kit (Hyglos
GmbH).
Results
Table 9 (below) shows the maximum LPS recoveries after unmasking as
dependent on the substance used as modulator. Furthermore, suitable
concentrations of stock solutions of the respective substances for
unmasking are shown.
TABLE-US-00009 TABLE 9 Optimum stock % LPS concentration of
Substances recovery substance X sodium octyl sulfate (SOS) 20 30 mM
1-decanoic acid 57 100 mM
As can be seen from the above, 1-alkyl alcohols are not the only
class of compounds which may function as a reconfiguring modulator
to promote the formation of a detectable form of LPS. Other
substances containing higher oxidation states of oxygen (e.g. as in
1-decanoic acid) as well as other heteroatoms than oxygen (e.g. as
in sodium octyl sulfate (SOS)) may also enable moderate to good
unmasking.
The results indicate that substances which are similar in structure
to 1-alkylalcohols are also able to support unmasking to a certain
extent. It appears that OH-derivatives of alkanes, preferably
C.sub.8-C.sub.16 alkanes, preferably C.sub.8-C.sub.12 alkanes,
preferably C.sub.12 alkanes serve best to render LPS susceptible to
detection by Factor C-based assays.
Example 9: Unmasking Using Albumins from Different Sources and
1-Dodecanol
As part of the verification of the improvement in unmasking by the
addition of bovine serum albumin (BSA) in masked samples containing
polysorbate 80, albumins from different sources were tested.
Materials and Methods
Masked samples (1 ml) containing 50 EU/ml of LPS (O55:B5) in
polysorbate 80/citrate buffer were unmasked by the addition of 100
.mu.l of stock solutions with different concentrations of albumins
(bovine serum albumin (BSA), very low endotoxin, Serva GmbH; human
serum albumin (HSA, recombinantly produced in Pichia pastoris
(Sigma Aldrich); and Ovalbumin (Ova), EndoGrade Ovalbumin, Hyglos
GmbH) and subsequent addition of 100 .mu.l of a 100 mM 1-dodecanol
stock solution). Concentrations of albumin stock solutions were
100, 33, 10, 3.3 and 1 mg/ml. Due to the lower solubility of
ovalbumin in water, a 100 mg/ml solution of ovalbumin was not
prepared.
LPS recoveries were calculated following determination of
detectable LPS content using the EndoLISA.RTM. kit (Hyglos GmbH).
For EndoLISA.RTM. measurements the unmasked samples were 1:10 and
1:100 diluted in endotoxin-free water and subsequently measured
according to the kit instructions.
Results
Table 10 (below) shows the unmasking efficiency from a polysorbate
80/citrate masking system, as dependent on albumins from different
sources.
TABLE-US-00010 TABLE 10 [stock solution] protein (mg/ml) % LPS
recovery BSA 100 66.0 33 46.2 10 38.1 3.3 28.2 1 30.9 HSA 100 42.3
33 94.5 10 151.6 3.3 40.4 1 34.3 ovalbumin -- nd 33 79.4 10 59.0
3.3 33.0 1 19.6 nd = no data
The data show that all albumins tested are able to support
unmasking from a polysorbate 80 masking system. Suitable final
concentrations in the unmasked samples are 10 mg/ml for BSA, 1
mg/ml for HSA and 3.3 mg/ml for ovalbumin. The differences in
optimum concentrations may result from different affinities of the
albumins to the detergent in the masked sample.
Example 10: The Effect of Various Chaotropic Salts on Unmasking
Efficiency
Unmasking using the combination of substances CaCl.sub.2 (agent
influencing hydrogen bonding), BSA (adsorbing modulator), SDS
(displacing modulator) and 1-dodecanol (reconfiguring modulator)
(this entire combination is referred to as "CBSD") has been shown
above to efficiently unmask LPS when masked by e.g. TRITON.TM.
(non-ionic surfactant) X-100. The present experiments investigate
the effect of the nature of the chaotropic salt (agent influencing
hydrogen bonding stability) on unmasking efficiency. To this end,
the following experiments employ salts of increasing chaotropic
properties: Na+, Mg.sup.2+ and Ca.sup.2+, in each case presented as
the corresponding chloride salt.
Materials and Methods
Endotoxin masking was performed as follows: 50 EU/ml of E. coli LPS
O55:B5 was masked by allowing it to incubate for 3 days at room
temperature in a 10 mM citrate buffer solution (pH 7.5) containing
0.05% TRITON.TM. (non-ionic surfactant) X-100. Here, TRITON.TM.
(non-ionic surfactant) X-100 functioned as the detergent
masker.
Unmasking of endotoxin was performed as follows: 300, 100, 30, 10,
3 and 1 .mu.l of either a 5 M sodium chloride (NaCl), 1 M magnesium
chloride (MgCl.sub.2) or 1 M calcium chloride (CaCl.sub.2) stock
solution were added to 1 ml aliquots of the masked samples and
mixed. Subsequently, 100 .mu.l of the other modulator components
(BSA (adsorbing modulator), SDS (disrupting and displacing
modulator) and 1-dodecanol (reconfiguring modulator)) were added as
described in Examples 1-5.
Results
Table 11 (below) shows the percentage of endotoxin recovery as
dependent on each chaotropic salt and the most suitable final
concentration of each salt in the unmasked sample.
TABLE-US-00011 TABLE 11 LPS recovery Concentration salt % (mM) NaCl
96.7 357 MgCl.sub.2 139.8 188 CaCl.sub.2 142.5 72
The data show that all the salts tested were able to support
efficient unmasking of LPS from the masking detergent TRITON.TM.
(non-ionic surfactant) X-100 in combination with a multicomponent
modulator system including BSA (as adsorbing modulator), SDS (here,
as disrupting modulator) and 1-dodecanol (as disrupting and
reconfiguring modulator). Furthermore, as described herein above,
the amount of the salt required to achieve a comparable degree of
unmasking efficiency decreased with increasing chaotropic
properties. These results allow several general conclusions to be
drawn. First, when using a salt to destabilize a masked complex
between endotoxin and endotoxin masker, the chaotropic character of
this salt is an important factor in achieving efficient unmasking.
Second, the amount of salt required to achieve efficient unmasking
will generally vary inversely with the chaotropic strength of the
salt employed.
Example 11: Unmasking of Endotoxin from Samples Containing
Detergent and Phosphate Buffer
Most formulations of drugs which contain a protein (e.g. antibody)
as an active pharmaceutical ingredient (API) contain either
non-ionic detergents like polysorbate 20 or 80 together buffered in
either citrate or phosphate. In such formulations, the detergent
concentration is usually above the respective detergent's critical
micellar concentration (CMC). Furthermore pH-values of such
formulattions are often adjusted in order to ensure optimum
stability of the API.
With the above in mind, the investigations set out in this Example
sought to investigate the influence of pH value on unmasking
efficiency. In order to approximate the conditions prevailing in
pharmaceutical formulations containing a protein API as closely as
possible, the detergents polysorbate 20 and polysorbate 80 were
used as endotoxin maskers, and the solutions were
phosphate-buffered. In view of the results described herein above,
unmasking was performed using a combination of CaCl.sub.2
(chaotropic salt as an agent which influences hydrogen bonding
stability), BSA (adsorbing modulator), SDS (here, as disrupting
modulator) and 1-dodecanol (disrupting and reconfiguring
modulator). As Ca.sup.2+ and PO.sub.4.sup.3- form non-soluble
calcium-phosphate complexes, the calcium chloride solution was
stabilized by addition of a two-fold molar excess of citrate, pH
7.5.
Materials and Methods
Masking of endotoxin was performed as follows: To 1 ml samples,
each containing 10 mM of phosphate buffer of various pH-values and
either 0.05% polysorbate 20 or polysorbate 80, were added 100 EU/ml
of E. coli LPS O55:B5. Masking was allowed to proceed by incubating
these solutions for 7 days at room temperature. LPS-containing
control samples of phosphate buffers lacking detergent were
prepared, incubated and measured in parallel to the masking
samples.
Unmasking of endotoxin was performed as follows: A combination of
CaCl.sub.2, BSA, SDS and 1-dodecanol was added to each of the
samples as described in previous examples. To avoid calcium
phosphate precipitation and to adjust the pH of the samples, a
two-fold molar excess of citrate buffer pH 7.5 was added to each
sample before addition of the unmasking components.
Endotoxin content of the masked samples was determined using the
EndoZyme.RTM. kit of Hyglos GmbH at time zero, and after 7 days.
Endotoxin content of the unmasked samples was analyzed using the
EndoLISA.RTM. kit of Hyglos GmbH. The percentage of LPS recovery
after 7 days of masking and after unmasking was calculated in
reference to control samples at time zero.
Results
Table 12 (below) and FIGS. 10 and 11 show the percentage of LPS
recovery after 7 days of masking as dependent on the pH-value and
the percentage of LPS recovery after unmasking of the masked
samples.
TABLE-US-00012 TABLE 12 Polysorbate 20 masker Polysorbate 80 masker
phosphate recovery recovery recovery recovery buffer after after
after after (pH-value) masking [%] unmasking [%] masking [%]
unmasking [%] 1.6 81 143 104 188 2.8 146 150 179 189 4.0 156 305
130 206 5.8 4 158 27 237 7.0 1 160 0 221 8.9 0 156 0 187 12.1 3 192
1 128
The data show that masking in phosphate buffer solutions containing
detergent is strongly pH dependent. At pH values below 4, no
masking occurs after one week of sample incubation. At pH values
above 4 a strong masking effect is seen, resulting in detectable
LPS recoveries less than 1%.
The data also show conclusively that the unmasking approach
implemented renders the previously masked, undetectable LPS
detectable. Independent of the pH-value and the extent of masking,
100% or more of LPS can be recovered, i.e. detected.
Example 12: Unmasking Using Other Displacing Modulators than
SDS
As shown in the examples above, a combination of
CaCl.sub.2/BSA/SDS/1-doedecanol efficiently unmasked endotoxin
which is masked by TRITON.TM. (non-ionic surfactant) X-100
detergent. Several of the experiments described above suggests the
importance of including SDS in this scheme to achieve efficient
unmasking. The aim of the experiments described in the present
example is to investigate whether the modulator component SDS
(here, as disrupting modulator) can be exchanged for another
detergent without negatively impacting the unmasking effect
observed using SDS.
Materials and Methods
Masking of endotoxin was performed as follows: 1 ml aliquots of 10
mM citrate pH 7.5 containing 0.05% TRITON.TM. (non-ionic
surfactant) X-100 were prepared in endotoxin-free glass test tubes.
Subsequently, 10 .mu.l of a 10,000 EU/ml stock solution of LPS (LPS
055 B5, Sigma L2637-5MG) were added, vortexed for 1 min and stored
at room temperature for at least 24 hours. A positive LPS control
in water was prepared as follows: 10 .mu.l of a 10,000 EU/ml LPS
stock solution was added to 1 ml of endotoxin-free water, mixed and
identically incubated as the masking preparations. Further details
regarding the positive LPS-water control are indicated in Example
1.
Unmasking of endotoxin was performed as follows: To masked
solutions of LPS, prepared as indicated above, CaCl.sub.2, BSA,
detergent X and 1-dodecanol were added as described in the previous
examples, where "detergent X" (disrupting modulator) was varied in
identity and concentration. The following detergents were tested:
dioctyl sulfosuccinate sodium salt (AOT), sodium dodecyl benzene
sulfonate (SDBS), polyethylene glycol 4-nonylphenyl-3-sulfopropyl
ether potassium salt (PENS) and p-xylene-2-sulfonic acid hydrate
(XSA). Unmasking was performed as described in above examples,
endotoxin content was determined using the EndoLISA.RTM. kit of
Hyglos GmbH, and the percentage of LPS recovery was calculated with
reference to the LPS-water positive control. Further details
regarding the LPS-water positive control are described in Example 1
above.
Results
Table 13 shows the percentage of LPS recovery after unmasking using
detergents other than SDS in the CaCl.sub.2/BSA/[detergent
X]/1-dodecanol unmasking approach.
TABLE-US-00013 TABLE 13 Concentration LPS recovery Detergent
optimum [%] AOT 0.01% 24 SDBS 0.01% 34 PENS 0.10% 23 XSA 0.05%
26
The data show that other detergents besides SDS are able to support
unmasking as a disrupting modulator in a CaCl.sub.2/BSA/[detergent
X]/1-dodecanol unmasking approach. Furthermore, in the absence of
1-dodecanol no detergent was able to unmask LPS from TRITON.TM.
(non-ionic surfactant) X-100. As mentioned above, this suggests
that 1-dodecanol may play an important role (at least) as a
reconfiguring modulator which may be crucial for mediating the
transition of endotoxin from a solubilized (undetectable) to an
aggregated (detectable) state.
Example 13: Unmasking from Buffered Antibody Compositions as
Dependent on the Masking Detergent
The most commonly used formulations of protein-based drug products
contain phosphate buffer and non-ionic detergents such as
polysorbate 20 or polysorbate 80. Further, antibodies constitute
one of the most commonly formulated pharmaceutical protein
products. With this in mind, we sought to confirm whether the above
unmasking approaches for detergents- or protein-masking systems are
suitable for unmasking endotoxin in systems containing both
detergent and protein, where the protein is an antibody buffered in
phosphate. Polysorbate 20 and 80 were chosen as masking detergents
in these experiments because these two detergents are the most
commonly used detergents in protein drug formulations.
Materials and Methods
Endotoxin masking was performed as follows: 50 EU/ml of endotoxin
(E. coli O55:B5; Sigma L2637-5MG) was added to 1 ml aliquots of an
antibody solution containing 10 mg/ml of a bovine polyclonal IgG
antibody preparation, dissolved in 10 mM sodium phosphate pH 7.5
and 50 mM NaCl. Subsequently, either polysorbate 20 or polysorbate
80 were added to a final concentration of 0.05%, and the solutions
were incubated for 3 days at room temperature to allow masking to
occur. Further, controls containing the buffer solution without
detergent or antibody, as well as the buffer solution containing
either the antibody or the respective polysorbate were prepared and
treated like the masking samples. Each of the controls contained
the same amount of LPS.
Unmasking was performed as follows: Unmasking was performed by
addition of either 1-dodecanol or BSA/1-dodecanol or
CaCl.sub.2/BSA/SDS/1-dodecanol. 100 .mu.l of the following stock
solutions were added to 1 ml of sample solution: CaCl.sub.2 (1 M),
BSA (100 mg/ml), SDS (1%) and 1-dodecanol (100, 10 or 1 mM).
Furthermore, before addition of calcium chloride to a sample, the
sample was stabilized against calcium phosphate precipitation by
the addition of a final concentration of 200 mM sodium citrate pH
7.5. All stock solutions were added sequentially with two-minute
mixing steps following each addition. After addition and mixing of
the last component the samples were incubated for at least 30
minutes at room temperature. Afterwards, the samples were diluted
1:10 and 1:100 in endotoxin-free water and analyzed for endotoxin
content using the EndoLISA kit (Hyglos GmbH). The percentage of LPS
recovery was calculated with reference to the determined endotoxin
content in the buffer control (discussed in more detail in Example
1).
Results
Table 14a (below) shows the percentage of LPS recovery of the water
control, the buffer without detergent, the buffer containing
antibody or detergent and the buffer containing antibody and
detergent after 3 days of incubation at room temperature.
TABLE-US-00014 TABLE 14a polysorbate polysorbate 20 80 LPS re- LPS
re- sample type ingredients covery (%) covery (%) water control
water 100 100 buffer buffer without detergent 102 99 masking
control buffer + antibody 31 44 masking control buffer +
polysorbate 0 2 masking control buffer + polysorbate + 0 9
antibody
Table 14b (below) shows the percentage of LPS recovery from an
antibody solution after unmasking containing either polysorbate 20
or 80. Furthermore, it shows the concentrations of the added stock
solutions.
TABLE-US-00015 TABLE 14b polysorbate polysorbate 20 80 [CaCl2]
[BSA] [SDS] [1-Dodecanol] LPS re- LPS re- (M) (mg/ml) (%) (mM)
covery (%) covery (%) -- -- -- 100 16.6 9.1 -- -- -- 10 19.9 6.8 --
-- -- 1 0.0 5.0 -- 100 -- 100 40.8 11.2 -- 100 -- 10 2.6 6.3 -- 100
-- 1 1.6 11.5 1 100 1 100 4.8 3.0 1 100 1 10 15.9 23.1 1 100 1 1
67.3 90.8
The data show that the buffer solutions without polysorbate 20 or
80 do not mask the added LPS. The buffer solutions containing
antibody but no polysorbate mask .about.55% to 70% of the LPS,
suggesting that the antibody protein contributes a masking effect
of its own. The LPS recoveries from buffer solutions containing
polysorbate or polysorbate and antibody are below 10% when no
unmasking measures are taken. Thus, not only the detergent but also
the antibody is responsible for masking of LPS.
LPS recoveries after unmasking from the masking complexes
containing LPS, detergent and antibody are low using 1-dodecanol
alone (9 and 17% for polysorbate 80 and 20, respectively). Using a
combination of BSA (adsorbing modulator) and 1-dodecanol
(disrupting and reconfiguring modulator) allowed moderate LPS
recoveries of 11 and 41% for polysorbate 80 and 20, respectively.
Unmasking using a combination of CaCl.sub.2, BSA (adsorbing
modulator), SDS (displacing modulator) and 1-dodecanol (disrupting
and reconfiguring modulator), results in recoveries of 67% and 91%
of the masked LPS for polysorbate 20 and 80, respectively.
Interestingly, unmasking was achieved using a 1-dodecanol stock
solution with a concentration as low as 1 mM. Furthermore, in
contrast to the unmasking from detergent systems lacking protein,
using 1-dodecanol (disrupting and reconfiguring modulator) or BSA
(adsorbing modulator) and 1-dodecanol (disrupting and reconfiguring
modulator) do not unmask with greater efficiency than 50%. As shown
for lysozyme above, efficient unmasking was only be achieved in the
presence of CaCl.sub.2, BSA, SDS and 1-dodecanol.
Example 14: Unmasking from Compositions Containing Antibody and
Polysorbate 20 as Dependent on the Buffer Substance
It was determined in above Example 14 that the inventive unmasking
approaches described herein are suitable for unmasking compositions
which contain both detergent and buffered protein (antibody). In
view of this, it was then desired to investigate the influence of
buffer on unmasking efficiency. To this end, we chose 10 mM citrate
or 10 mM phosphate buffer of pH 7.5, because these are the most
commonly used buffers in protein drug formulations.
Materials and Methods
Endotoxin masking was performed as follows: 50 EU/ml of endotoxin
(E. coli O55:B5; Sigma L2637-5MG) were added to 1 ml aliquots of an
antibody solution containing 10 mg/ml of a bovine polyclonal IgG
antibody preparation, dissolved in either 10 mM sodium phosphate
containing 50 mM sodium chloride or 10 mM sodium citrate pH 7.5
containing 150 mM sodium chloride. Subsequently, polysorbate 20 was
added to a final concentration of 0.05% and samples were masked for
3 days at room temperature. Further, positive controls containing
the buffer solution without detergent or antibody, as well as the
buffer solution containing either the antibody or the respective
polysorbate were prepared and treated like the masking samples.
Each of the positive controls contained the same amount of LPS.
Endotoxin unmasking was performed as follows: Unmasking was
performed by addition of either 1-dodecanol or a combination of BSA
(adsorbing modulator) and 1-dodecanol (disrupting and reconfiguring
modulator) or CaCl.sub.2, BSA (adsorbing modulator), SDS
(displacing modulator) and 1-dodecanol (disrupting and
reconfiguring modulator). 100 .mu.l of each of the following stock
solutions were sequentially added to 1 ml of sample solution:
CaCl.sub.2 (1M), BSA (10 mg/ml), SDS (1%) and 1-dodecanol (100, 10
or 1 mM). Furthermore, before addition of calcium chloride to a
phosphate buffer-containing sample, this sample was stabilized
against calcium phosphate precipitation by the addition of a final
concentration of 200 mM sodium citrate pH 7.5. All stock solutions
were added sequentially with two-minute mixing steps after each
addition. After addition and mixing of the last component the
samples were incubated for at least 30 minutes at room temperature.
Afterwards, the samples were diluted 1:10 and 1:100 in
endotoxin-free water and analyzed for endotoxin content using the
EndoLISA kit (Hyglos GmbH). The percentage of LPS recovery was
calculated with reference to the determined endotoxin content in
the positive control (discussed in more detail in Example 1).
Results
Table 15 (below) shows the percentage of LPS recovery from an
antibody solution after masking and unmasking containing either
citrate or phosphate as buffer substance.
TABLE-US-00016 TABLE 15 citrate phosphate buffer buffer LPS LPS
sample type ingredient recovery (%) recovery (%) water control
water 100 100 masking control buffer + antibody 40 31 masking
control buffer + polysorbate 20 0 0 masking control buffer +
polysorbate 20 + 1 0 antibody LPS [1- LPS [1- unmasking re- dodec-
re- dodec- sample approach/ covery anol] covery anol] type
ingredients (%) (mM) (%) (mM) unmasked 1-dodecanol 26 100 17 100
sample * unmasked BSA/ 49 100 41 100 sample * 1-dodecanol unmasked
CaCl.sub.2/ 87 100 67 1 sample * BSA/SDS/ 1-dodecanol * "Unmasked"
samples contained antibody.
The data show that the buffer solutions containing antibody but no
polysorbate, mask 60 to 70% of the LPS (based on the recovery of
40% and about 30% LPS for citrate and phosphate buffers,
respectively). The LPS recoveries from buffer solutions containing
polysorbate or polysorbate and antibody are below 1%. In these
cases, masking is independent of the buffer present.
LPS recoveries after unmasking from the compositions containing
LPS, detergent and antibody are low using 1-dodecanol alone (17%
and 26% for phosphate and citrate, respectively) and moderate using
a combination of BSA (adsorbing modulator) and 1-dodecanol
(disrupting and reconfiguring modulator) (41% and 49% for phosphate
and citrate, respectively). Unmasking using a combination of
CaCl.sub.2, BSA (adsorbing modulator), SDS (displacing modulator
and 1-dodecanol (disrupting and reconfiguring modulator) results in
recoveries of 67% and 87% of the masked LPS for phosphate and
citrate, respectively. Interestingly, the necessary concentration
of 1-dodecanol stock solution for efficient unmasking differs
strongly between the buffer systems used (100 mM for
antibody/detergent/citrate and 1 mM for
antibody/detergent/phosphate). The data clearly show that efficient
unmasking of endotoxin in compositions comprising both protein
(antibody) and detergent can be achieved by adjustment of
1-dodecanol concentration.
Example 15: Masking and Unmasking of an Antibody Solution
Containing LPS from Unknown Source
To show that unmasking is not only possible from solutions
containing LPS from a known source, we tested a commercially
available mouse monoclonal antibody for diagnostic use which
contains an LPS contamination, where the source of the LPS is
unknown. Furthermore, this antibody was dissolved in a buffer
composition which corresponds to the formulation of the known
antibody drug product Rituximab (MabThera.RTM., Rituxan.RTM.).
Materials and Methods
Determination of endotoxin contamination: A mouse monoclonal
antibody (MAB 33, Roche Diagnostics) was dissolved in a solution
containing citrate and sodium chloride of pH 6.5 and stored at
4.degree. C. The final concentrations of citrate, sodium chloride
and antibody were 25 mM, 150 mM and 10 mg/ml, respectively.
Directly after solubilization of the antibody, the endotoxin
content was analyzed using EndoZyme.RTM. and EndoLISA.RTM.
detection kits (Hyglos GmbH). The determined endotoxin content was
11 EU/mg of antibody.
LPS masking was initiated by addition of polysorbate 80 to a final
concentration of 0.07% and increasing the temperature to ambient
conditions (22.degree. C.). Afterwards, 1 ml aliquots of the
samples were incubated at room temperature for 3 days to allow the
endottoxin present to become masked.
Unmasking was performed as follows: Unmasking was performed by
addition of either 1-dodecanol (disrupting and reconfiguring
modulator); or a combination of BSA (adsorbing modulator) and
1-dodecanol (disrupting and reconfiguring modulator); or a
combination of CaCl.sub.2, BSA (adsorbing modulator), SDS
(displacing modulator) and 1-dodecanol (disrupting and
reconfiguring modulator). 100 .mu.l of each of the following stock
solutions were sequentially added to 1 ml of sample solution:
CaCl.sub.2 (1 M), BSA (10 mg/ml), SDS (1%) and 1-dodecanol (100, 10
or 1 mM). All stock solutions were added sequentially with
two-minute mixing steps after each addition. After addition and
mixing of the last component the samples were incubated for at
least 30 minutes at room temperature.
Afterwards, the samples were diluted 1:10 and 1:100 in endotoxin
free water and analyzed for endotoxin content using EndoLISA.RTM.
(Hyglos GmbH). The percentage of LPS recovery was calculated in
reference to the determined endotoxin content at time zero.
Results
Table 16 (below) shows the percentage of endotoxin recovery as
dependent on the masking time, the presence or absence of
polysorbate 80 and unmasking from antibody/polysorbate 80
solution.
TABLE-US-00017 TABLE 16 LPS recovery sample type ingredients (%)
control t(0) buffer + antibody 100 masking control (3 days) buffer
+ antibody 57 masking control (3 days) buffer + polysorbate 80 0
masking control (3 days) buffer + polysorbate 80 + 3 antibody LPS
[1- unmasking approach/ recovery dodecanol] sample type ingredients
(%) (mM) unmasked sample * 1-dodecanol 45 100 unmasked sample *
BSA/1-dodecanol 68 100 * "Unmasked" samples contained antibody.
The data show that the buffer solution containing antibody but no
polysorbate masks 40% of the LPS within 3 days of incubation at
room temperature. However, incubation in buffer containing either
polysorbate 80 or antibody and polysorbate 80, results in endotoxin
recoveries smaller than 4%.
Unmasking from the antibody/detergent samples results in recoveries
of 45% using 1-dodecanol (disrupting and reconfiguring modulator);
68% using a combination of BSA (adsorbing modulator) and
1-dodecanol (disrupting and reconfiguring modulator); and 179%
using a combination of CaCl.sub.2, BSA (adsorbing modulator), SDS
(displacing modulator) and 1-dodecanol (disrupting and
reconfiguring modulator). In the latter case, the best recovery is
achieved using a 1 mM 1-dodecanol stock solution.
The experiments described in this example show that, when present,
naturally occurring endotoxin (NOE) can be detected by a suitable
endotoxin detection system. Furthermore, these experiments show
that such NOE can be masked in the manner described herein above,
i.e. the danger of masking applies not only for purified endotoxin,
but also for NOE. The ability of the inventive methods as described
herein to unmask such NOE further demonstrate their applicability
to situations in which NOE has been masked, proving their
effectiveness of the inventive methods to unmask masked NOE. These
findings are relevant to the conditions prevailing in industry,
where production processes often start with an expressed protein in
the presence of NOE, and the latter is masked by incorporation of
detergent to prevent unwanted protein aggregation. Overall, then,
the results of the experiments described in this example
demonstrate that the inventive methods are able to unmask endotoxin
under conditions of relevance for the pharmaceutical industry.
These data also clearly show that unmasking is independent of the
source and purity of the LPS.
In all three cases of masking in antibody solutions (Examples 13,
14 and 15), it can be seen that masking is not only due to the
detergent component in the composition but also to some extent to
the antibody itself. The most efficient unmasking approach is to
use a combination of CaCl.sub.2, BSA (adsorbing modulator), SDS
(displacing modulator) and 1-dodecanol (disrupting and
reconfiguring modulator) to unmask the endotoxin. Here, analogies
can be seen to the lysozyme case (discussed in Example 7 above), in
which the protein itself plays a role as an endotoxin masker.
Interestingly, in all cases, the concentration of 1-dodecanol
should be optimized for efficient unmasking.
Example 16: General Evaluation of Unmasking Approach as Applied to
a New Composition in Question
As shown in the above examples, the choice of the approach taken to
unmask endotoxin suspected of being present, but masked in a
composition will depend on a number of factors. For instance, as
the foregoing examples have shown, it is sometimes possible to
achieve efficient unmasking using a single-component modulator
which doubles as a disrupting modulator and a reconfiguring
modulator, as defined herein above. On the other hand, in some
instances, the modulator should be a modulator system with two or
more components, for instance a displacing modulator and/or an
adsorbing modulator, depending on what measures are needed to
destabilize and disrupt the endotoxin/endotoxin masker complex
sufficiently such that the endotoxin is liberated and can be
mediated into an aggregated form which can be detected.
The above examples start from known, controlled solution conditions
in order to illustrate concepts underlying the present invention.
In a real-world scenario, however, in which the methods of the
invention are to be applied to a new composition in question, it is
necessary to first evaluate the approach of the methods of the
invention before meaningful results can be obtained. The present
example addresses such a validation, setting out a generic scheme
by which the methods of the invention may be calibrated to a new
composition in question. To this end, an iterative unmasking
approach is necessary, starting with an initial screening for the
best suited unmasking approach followed by subsequent improvement
steps for adjustment of optimum unmasking component
concentrations.
General Description of an Evaluation Process for a Given
Composition
Generally, FIG. 12 shows a scheme which schematically sets out the
steps which one would normally take in evaluating the inventive
methods for a new, unknown composition.
As will be clear from the above, ultimate detection of initially
masked endotoxin depends on the ability to convert this endotoxin
from stably bound (masked) form to an aggregated from which is
unmasked and therefore detectable. The component of the modulator
responsible for this final conversion is the reconfiguring
modulator. The first step of FIG. 12 reflects this, in that it
specifies a first step of determining an optimal concentration of
reconfiguring modulator (e.g. 1-dodecanol). Step 2 then optimizes
the concentration of adsorbing modulator, if this modulator is
included. Step 3 then optimizes the concentration of displacing
modulator, if this modulator is included.
It should be emphasized that not all three steps will always be
needed. If one already sees that a composition, for example a
pharmaceutical composition, in question contains significant
amounts of endotoxin following step one, then this answer may
already be enough to conclude that the composition thought to be
endotoxin-free was really not.
Specific Description of Evaluation Process for a Given
Composition
FIG. 13 shows the combinations and concentrations of stock
solutions for selecting and optimizing the unmasking process. The
unmasking approaches are divided into different possible scenarios
A, B and C, depending on which substance or combination of
substances is/are used in unmasking. Unmasking approach A describes
an unmasking approach in which only 1-dodecanol is used as a
modulator. Unmasking approach B describes an unmasking approach in
which the modulator system is composed of 1-dodecanol and BSA.
Unmasking approach C describes an unmasking approach in which the
modulator system is composed of 1-dodecanol, BSA and SDS, and is
performed in the presence of CaCl.sub.2.
Procedure
Add 100 .mu.l of the unmasking component stock solutions to 1 ml of
masked sample. After addition of one component, mix sample
thoroughly by vortexing for 2 minutes. Then, add the next component
and mix. After addition of all components and subsequent mixing,
incubate samples for >30 minutes at room temperature.
Afterwards, analyze samples for endotoxin content using an
appropriate endotoxin testing method, e.g. the EndoLISA.RTM. kit of
Hyglos GmbH.
Example 17: Detection of Unmasked Endotoxin Using a Recombinant
Factor C Assay
This experiment investigates the effect of unmasking endotoxin
using a multi-component modulator comprising CaCl.sub.2, BSA, SDS
and dodecanol. Endotoxin content of the masked and unmasked samples
was determined using the EndoZyme.RTM. kit of Hyglos GmbH. The
experiment was performed in order to show that detection of
unmasked endotoxin can be achieved using different detection
assays.
Materials and Methods
Endotoxin (E. coli O55:B5, Sigma L2637-5MG) was masked in solutions
containing 1.times.PBS-buffered 0.05 wt % Polysorbate 80 or
1.times.PBS buffered 0.05 wt % Polysorbate 20 for 3 days at room
temperature.
Unmasking was performed as follows: Unmasking was performed by a
combination of sodium citrate, CaCl.sub.2, BSA, SDS and
1-dodecanol. 150 .mu.L of sodium citrate and 100 .mu.l of each of
the following stock solutions were added to 1 ml of sample
solution: sodium citrate (1.375 M pH 7.5), CaCl.sub.2 (1 M), BSA
(10 mg/ml), SDS (1%) and 1-dodecanol (1 mM). 1-dodecanol was
solubilized in 70% EtOH. In a separate masking control, no
unmasking was performed.
All stock solutions were added sequentially with two-minute mixing
steps after each addition. After addition and mixing of the last
component the samples were incubated for at least 30 minutes at
room temperature.
Subsequently, masked (masking control) and unmasked samples were
diluted stepwise 1:10 and 1:5 in depyrogenated water (final
dilution 1:50). A recombinant Factor C assay (EndoZyme.RTM.) was
used for detection of endotoxin.
Results
Table 17 (below) shows the percent recovery, measured using a
recombinant Factor C assay (EndoZyme.RTM.), of endotoxin recovered
from the two masking systems specified above in this example.
TABLE-US-00018 TABLE 17 Detection of unmasked endotoxin using
recombinant Factor C Recombinant Factor C PBS + P80 PBS + P20
Sample [EU/mL] [EU/mL] Positive control 9.3 6.8 Recovery [%]
Recovery [%] Masking control 0 0 After unmasking 65 66
The masking control showed no endotoxin recovery in either sample.
Unmasking of endotoxin in polysorbate 80 or polysorbate 20 resulted
in endotoxin recovery of 65% and 66%, respectively, with reference
to the positive control (endotoxin content in depyrogenated water).
The results indicate the efficient demasking of endotoxin using a
multi-component modulator comprising Sodium citrate, CaCl.sub.2,
BSA, SDS and dodecanol as detected by a recombinant Factor C
detection system (EndoZyme.RTM.). This experiment proves that the
detection of unmasked endotoxin is independent of the endotoxin
detection system used.
Accordingly, unmasked endotoxin may be detected using the endotoxin
detection system employed in previous examples, but may also be
detected using an endotoxin detection system differing from that
used in previous examples.
Example 18: Detection of Unmasked Endotoxin Using a Limulus
Ameboecyte Lysate (LAL) Assay
This experiment investigates the detection of unmasked endotoxin
using a detection assay different from the recombinant Factor C
assay (EndoZyme.RTM.), i.e. the Limulus Ameboecyte Lysate (LAL)
assay. The experiment was performed in order to further corroborate
that detection of endoxin unmasking does not depend on the
detection assay.
Materials and Methods
Endotoxin (E. coli O55:B5, Sigma L2637-5MG), was masked in
solutions containing 1.times.PBS-buffered 0.05 wt % Polysorbate 80
or 1.times.PBS buffered 0.05 wt % Polysorbate 20 for 3 days at room
temperature.
Unmasking was performed as follows: Unmasking was performed by a
combination of sodium citrate, CaCl.sub.2, BSA, SDS and
1-dodecanol. 150 .mu.L of sodium citrate and 100 .mu.l of each of
the following stock solutions were added to 1 ml of sample
solution: sodium citrate (1.375 M pH 7.5), CaCl.sub.2 (1 M), BSA
(10 mg/ml), SDS (1%) and 1-dodecanol (1 mM). 1-dodecanol was
solubilized in 70% EtOH.
All stock solutions were added sequentially with two-minute mixing
steps after each addition. After addition and mixing of the last
component the samples were incubated for at least 30 minutes at
room temperature.
Subsequently, masked (masking control) and unmasked samples were
diluted stepwise 1:10 and 1:5 in depyrogenated water (final
dilution 1:50). A kinetic LAL-based chromogenic assay
(kinetic-QCL.RTM., Lonza) was used for detection of endotoxin.
Masking control reflects the detectable endotoxin content without
unmasking. In a separate masking control, no unmasking was
performed.
Results
Table 18 (below) shows the percent recovery, measured using an LAL
assay (kinetic QCL.RTM., Lonza), of endotoxin recovered from the
two masking systems specified above in this example.
TABLE-US-00019 TABLE 18 Unmasking using an LAL assay LAL PBS + P80
PBS + P20 Sample [EU/mL] [EU/mL] Positive control 11.6 7.2 Recovery
[%] Recovery [%] Masking control 3 0 After unmasking 96 47
The masking control showed no endotoxin recovery in both samples.
Unmasking of endotoxin in polysorbate 80 or polysorbate 20 resulted
in endotoxin recovery of 96% and 47%, respectively, with reference
to the positive control (endotoxin content in depyrogenated water).
The data clearly demonstrate that unmasking of endotoxin can be
detected with the LAL detection assay and that detection of
endotoxin unmasking does not depend on the detection assay.
Example 19: Variation of Alkanols (Aliphatic Alcohols) as
Modulators for Unmasking Using a Multi-Component Modulator
This experiment investigates unmasking of different endotoxins
using different alkanols. The experiment was performed in order to
investigate the unmasking efficiency of different alkanol compounds
in the multi-component modulator.
Materials and Methods
Endotoxin from E. coli O55:B5 (Sigma L2637-5MG), S. abortus equi
(Acila 1220302) and K. pneumoniae (LMU) were masked in solutions
containing 10 mM sodium citrate and 0.05 wt % Polysorbate 20 for
three days at room temperature.
Unmasking was performed as follows: Unmasking was performed by a
combination of NaCitrate, CaCl.sub.2, BSA, SDS and 1-dodecanol. 150
.mu.L of sodium citrate and 100 .mu.l of each of the following
stock solutions were added to 1 ml of sample solution: sodium
citrate (1.375 M pH 7.5), CaCl.sub.2 (1 M), BSA (10 mg/ml), SDS
(1%) and a certain concentration of 1-dodecanol. The alkanols and
alkanol mixtures used in the multi-component modulator systems were
solubilized in EtOH; concentrations are listed in Table 19a
(below). In a separate masking control, no unmasking was
performed.
All stock solutions were added sequentially with two-minute mixing
steps after each addition. After addition and mixing of the last
component the samples were incubated for at least 30 minutes at
room temperature.
TABLE-US-00020 TABLE 19a Unmasking Concentration approach: Alkanols
(size) [mM] 1 Octanol (C8) 1.0 2 Decanol (C10) 1.0 3 Dodecanol
(C12) 1.0 4 Tetradecanol (C14) 1.0 5 Hexadecanol (C16) 1.0 6
Octanol (C8) 0.3 Decanol (C10) 0.3 Dodecanol (C12) 0.3 7 Decanol
(C10) 0.3 Dodecanol (C12) 0.3 Tetradecanol (C14) 0.3 8 Dodecanol
(C12) 0.3 Tetradecanol (C14) 0.3 Hexadecanol (C16) 0.3
Afterwards, the samples were diluted 1:10 and 1:100 in endotoxin
free water and analyzed for endotoxin content using EndoLISA.RTM.
(Hyglos GmbH). The percentage of LPS recovery was calculated in
reference to the determined endotoxin content at time zero
(summarized in Table 19b, below).
Results
Table 19b (below) shows the percent recovery after masking (masking
control) and after unmasking using the EndoLISA.RTM. assay (Hyglos)
from the above masking system by various unmasking approaches
employing different alkanols (aliphatic alcohols) or alkanol
mixtures (aliphatic alcohol mixtures) as specified above in Table
19a.
TABLE-US-00021 TABLE 19b Unmasking of different endotoxins using
Ca, BSA, SDS and varying alkanols, as detected by the EndoLISA
.RTM. assay Endotoxin K. pneumoniae * S. abortus equi E. coli
O55:B5 [EU/mL] [EU/mL] [EU/mL] Positive Control 191 51 68 Recovery
[%] Recovery [%] Recovery [%] Masking Control 0 0 0 Unmasking
approach (alkanol size) 1 (C8) 75 0 2 2 (C10) 52 0 0 3 (C12) 147 62
76 4 (C14) 94 108 71 5 (C16) 99 83 22 6 (C8, C10, C12) 60 14 6 7
(C10, C12, C14) 126 108 43 8 (C12, C14, C16) 126 173 43 * For
unmasking of K. pneumoniae 150 .mu.L of CaCl.sub.2 were added.
The above results indicate that unmasking of K. pneumoniae was
achieved with octanol (75% recovery), dodecanol (147%),
tetradecanol (94%) and hexadecanol (99%), as well as with different
combinations of alkanols (see e.g. unmasking approaches 7 and 8).
Unmasking with decanol, however, was less efficient (52%).
Unmasking of the S. abortus equi LPS was most efficient using
tetradecanol (108%), hexadecanol (82%), dodecanol (62%), or
different combinations of alkanols. Effective unmasking of E. coli
O55:B5 was observed for dodecanol (76%) and tetradodecanol (71%).
No endotoxin recovery was observed for the masking controls.
These results indicate that the most efficient unmasking
(independent of the nature of the endotoxin) was achieved using
dodecanol or tetradecanol, or using combinations of dodecanol and
tetradecanol with a further alkanol (e.g. decanol in demasking 7).
These results also indicate that all multi-component modulator
systems with C.sub.12, C.sub.14 and/or C.sub.16 aliphatic alcohols
exhibited efficient unmasking of endotoxin.
The range of alkyl chain length of the fatty alcohols for efficient
unmasking seems to depend on the endotoxin source. The differences
in the unmasking efficiencies may depend to a certain extent on the
heterogeneity in length of the acyl chains of the
.beta.-hydroxy-fatty acids which are present in the Lipid A portion
of endotoxin. Between and within bacterial species, these acyl
chains can vary in length from C.sub.10 to C.sub.28 (Endotoxin in
health and disease, edited by H. Brade (1999), p98 et seq:
"Chemical structure of Lipid A: Recent advances in structural
analysis of biologically active molecules"; Marcel Dekker Inc, New
York). However, most commonly .beta.-hydroxy-fatty acids with
chains length of C14 and C16 are appended to the diglucosamine of
Lipid A. Thus, unmasking is in all cases most efficient in the
presence of fatty alcohols with alkyl chain length between C12 and
C14, although unmasking of endotoxin is also observed for other
alkyl chain lengths in the C8-C16 range.
Example 20: Variation of Alkanols (Aliphatic Alcohols) as
Modulators for Unmasking Using a Single-Component Modulator
This experiment was performed to investigate the effect of various
alkanols (aliphatic alcohols) on unmasking in the absence of
additional modulator components. The experiment thus investigates
the efficiency of endotoxin unmasking using various alkanols
(aliphatic alcohols) as single-component modulators.
Materials and Methods
Endotoxin E. coli O55:B5 (Sigma L2637-5MG) was masked in solutions
containing 10 mM sodium citrate and 0.05 wt % Polysorbate 20 for 3
days at room temperature.
In order to unmask the samples, samples (1 mL) were mixed with 100
.mu.L of the particular alkanol (i.e. aliphatic alcohol). The
alkanols used in the single-component modulator systems were
solubilized in EtOH. Concentrations are shown in Table 20a
(below).
TABLE-US-00022 TABLE 20a Variation of alkanols (aliphatic alcohols)
Unmasking Approach Alkanols (size) Concentration [mM] 1 Dodecanol
(C12) 50 mM 2 Tridecanol (C13) 50 mM 3 Tetradecanol (C14) 50 mM
After addition of unmasking agents, the samples were incubated for
30 minutes and diluted 1:10 as well as 1:100 in depyrogenated
water. Endotoxin was detected in both dilutions and the stated
recovery reflects the mean recovery of both dilutions. The masking
control reflects the non-treated sample after masking, i.e. the
solution is not unmasked. The EndoLISA.RTM. assay was used for
endotoxin detection.
Results
Table 20b (below) shows the percent recovery, measured using the
EndoLISA.RTM. assay (Hyglos), of endotoxin recovered from the above
masking system by various unmasking approaches employing different
alkanols (aliphatic alcohols) in different unmasking approaches
using single-modulator systems as specified above in Table 20a.
TABLE-US-00023 TABLE 20b Unmasking using different alkanols
(EndoLISA .RTM.) E. coli O55:B5 (gel) Endotoxin [EU/mL] Positive
Control 111 Recovery [%] Masking Control 0 Unmasking approach
(alkanol size) 1 (C12) 56 2 (C13) 41 3 (C14) 22.6
The results indicate that a single-component modulator consisting
of dodecanol (unmasking approach 1) was most efficient in unmasking
of E. coli O55:B5 (56% recovery), whereas single-component
modulators consisting of tridecanol (unmasking approach 2) or
tetradecanol (unmasking approach 3) resulted in less recovery of E.
coli O55:B5 (41% and 22.6%, respectively). As expected, the masking
controls showed no endotoxin recovery. In summary, the data
demonstrate that the most efficient alkanol (aliphatic alcohol) for
unmasking of E. coli O55:B5, when used as a single-component
modulator system, is dodecanol, followed by tridecanol and
tetradecanol.
* * * * *
References